WO2011149305A9 - Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction - Google Patents

Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction Download PDF

Info

Publication number
WO2011149305A9
WO2011149305A9 PCT/KR2011/003903 KR2011003903W WO2011149305A9 WO 2011149305 A9 WO2011149305 A9 WO 2011149305A9 KR 2011003903 W KR2011003903 W KR 2011003903W WO 2011149305 A9 WO2011149305 A9 WO 2011149305A9
Authority
WO
WIPO (PCT)
Prior art keywords
seq
probe
primer
tuberculosis
mycobacterium tuberculosis
Prior art date
Application number
PCT/KR2011/003903
Other languages
French (fr)
Korean (ko)
Other versions
WO2011149305A3 (en
WO2011149305A2 (en
Inventor
김정욱
Original Assignee
울산대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020110009530A external-priority patent/KR101158649B1/en
Application filed by 울산대학교 산학협력단 filed Critical 울산대학교 산학협력단
Priority to US13/700,157 priority Critical patent/US20130210005A1/en
Priority to CN201180036998.5A priority patent/CN103038348B/en
Publication of WO2011149305A2 publication Critical patent/WO2011149305A2/en
Publication of WO2011149305A9 publication Critical patent/WO2011149305A9/en
Publication of WO2011149305A3 publication Critical patent/WO2011149305A3/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a method for detecting Mycobacterium tuberculosis bacteria and anti-acidic non-tuberculosis bacteria. More specifically, the tube set and / or probes for detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria that can detect the gene sequence specific to the tuberculosis bacteria and acidic non-TB tuberculosis, tuberculosis bacteria and acidic non-TB tuberculosis detection kit and dual real-time polymerization using the same The present invention relates to a method for simultaneously detecting Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis using enzyme chain reaction.
  • Nontuberculous mycobacteria have been recognized as non-pathogenic bacteria widely present in natural environments such as soil and water.
  • AIDS has been prevalent, and it has been confirmed that the bacterium is an opportunistic strain of M. tuberculosis as an opportunistic strain of patients with AIDS, and that it can cause infection in normal patients. Awareness has spread.
  • tuberculosis smears and culture-positive specimens are isolated from non-acidic non-tuberculosis bacteria, and in Japan, Hong Kong and Korea, about 10-20% of the anti-acidic tuberculosis bacteria isolated from sputum cause lung disease and In the United States, Canada and Western Europe, about 40-50% are known to cause lung disease.
  • pulmonary disease caused by acidic non-tuberculosis bacillus is easy to be misdiagnosed because it is similar to the slowly progressing pulmonary tuberculosis.
  • the drugs showing susceptibility to tuberculosis bacteria and anti-acidic non-tuberculosis bacillus are different, the rapid and accurate method of separating and detecting tuberculosis bacteria and acidic non-tuberculosis bacillus Is being requested.
  • test reagents for detecting tuberculosis bacteria and non-acidic non-tuberculosis bacteria are not unique to acidic non-tuberculosis bacteria.
  • the problem of poor detection and diagnosis accuracy is that it is incorrectly identified as an acidic non-tuberculosis bacterium that reacts only to the primers of an acidic non-tuberculosis bacterium without reacting to the primer, or when only an acidic non-tuberculosis bacillus is detected when both the acidic non-tuberculosis bacillus and tuberculosis bacteria are present. It is happening.
  • An object of the present invention is to provide a primer set having a high detection ability in Mycobacterium tuberculosis-specific IS6110 gene for accurate detection and diagnosis of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis, and a primer set having high detection ability in 16S rRNA gene of mycobacterium tuberculosis.
  • Another object of the present invention is to provide a detection kit of Mycobacterium tuberculosis and anti-acidic Mycobacterium tuberculosis comprising the primer set and / or probe.
  • the present invention provides a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 9.
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 1 and a reverse primer of nucleotide sequence 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And it provides a Mycobacterium tuberculosis bacillus and anti-acidic Mycobacterium tuberculosis detection kit comprising a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9.
  • the present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-tuberculosis bacterium 16S rRNA gene of SEQ ID NO: 9; And it provides a method for detecting Mycobacterium tuber
  • the present invention is a forward primer of SEQ ID NO: 22; One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And it provides a primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
  • the present invention provides a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 23.
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And it provides a tuberculosis bacteria and anti-acidic tuberculosis bacteria detection kit comprising a probe for detecting the non-acidic tuberculosis 16S rRNA gene of SEQ ID NO: 23.
  • the present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primers specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 23; And it provides a method for detecting Mycobacterium tuberculosis and
  • the present invention provides a probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26.
  • the present invention provides a probe for detecting non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28.
  • the present invention provides a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic tuberculosis 16S rRNA gene comprising a probe of the base sequence 27 and the probe of the base sequence 28.
  • the present invention comprises the steps of separating the DNA from the sample;
  • a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25;
  • it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
  • the present invention provides a probe for detecting non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39.
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for detecting the anti-acidic tuberculosis 16S rRNA gene selected from the group consisting of a probe of the nucleotide sequence 37, the probe of the nucleotide sequence 38 and the nucleotide sequence 39.
  • the present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria compris
  • the present invention is a forward primer of SEQ ID NO: 61; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer comprising a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 8.
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63.
  • the present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 62 or a probe of SEQ ID NO: 63; And it provides a method for detecting Myco
  • the present invention provides a primer comprising a primer of nucleotide sequence 65, a primer of nucleotide sequence 66 and a primer of nucleotide sequence 67; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of nucleotide sequence 36.
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the probe of SEQ ID NO: 39 or the base sequence 68.
  • the present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 39 or a probe of SEQ ID NO: 68; And it provides a method for detecting Mycobacterium tuberculosis and non
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76.
  • the present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
  • the present invention provides a primer set and / or a probe for detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis, which can detect gene sequences specific for Mycobacterium tuberculosis and Mycobacterium tuberculosis, and a detection kit comprising the same and a dual real-time polymerase chain reaction method using the same. It is possible to provide a method for detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria.
  • 1 and 2 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in the green and yellow channels of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 3 and 4 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
  • 5 and 6 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
  • FIG 7 and 8 are graphs showing the change in the fluorescence intensity of the cycle number of the polymerase chain reaction in the yellow channel and the green channel of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 9 and 10 are graphs showing the change in fluorescence intensity for the number of cycles of the polymerase chain reaction in the yellow channel and the green channel of the double-real-time polymerase chain reaction of NRT.
  • 11 and 12 show y-axis for the number of cycles of polymerase chain reaction in the yellow channel and the green channel of the double real-time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • FIG. 13 and 14 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 15 and 16 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
  • 17 and 18 are y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (MTC) + acidic non-TB tuberculosis (NTM), respectively, indicating a change in fluorescence intensity. It is a graph.
  • 19 and 20 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 21 and 22 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
  • FIG. 23 and FIG. 24 show y-axis for the number of cycles of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • 25 and 26 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • FIG. 27 and FIG. 28 are graphs showing changes in fluorescence intensity of the cycle number of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction of NRT.
  • 29 and 30 show y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It's a graph.
  • 31 and 32 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 33 and 34 are graphs showing changes in fluorescence intensity of cycles of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction (NTM).
  • 35 and 36 show the y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
  • FIG. 37 and 38 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 39 and 40 are graphs showing changes in fluorescence intensity of the cycle number of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction (NTM).
  • 41 and 42 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • the present invention provides a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9.
  • the 5 ′ end of the probe is one fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED.
  • Labeled with a factor, and the 3 ′ end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and molecular grove binding non-fluorescence quencher (MBGBFQ).
  • Probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9 is a forward primer of SEQ ID NO: 4; One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And it is a probe specific to the reaction product of the polymerase chain reaction method using a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
  • the present invention provides a primer set specific to the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 1 and a reverse primer of nucleotide sequence 2;
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 9 and the Mycobacterium tuberculosis IS6110 gene detection probe of SEQ ID NO: 3 may be labeled with different detectable means.
  • the detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
  • the Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 1 and a reverse primer of nucleotide sequence 2.
  • the primer set of the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes ( MTCs ).
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3 and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 9 may be a Taqman probe.
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3 is specific for the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 Probe.
  • the forward primer of SEQ ID NO: 4 is 16S rRNA gene specific forward primer of non-acidic tuberculosis bacteria.
  • nucleotide sequence 4 5′-ggyrayctgccctgcac-3 ′
  • 5′-ggtaatctgccctgcac-3 ′ base sequence 12
  • 5′-ggtaacctgccctgcac-3 ′ base sequence 13
  • 5′-ggcaatctgccctgcac-3 ′ SEQ ID NO: 14
  • 5′-ggcaacctgccctgcac-3 ′ SEQ ID NO: 15
  • 5′-ggtgatctgccctgcac-3 ′ SEQ ID NO: 16
  • 5′-ggtgacctgccctgcac-3 ′ SEQ ID NO: 17
  • 5′-ggcgatctgccctgcac-3 Primer set comprising ′ (base 18) and 5
  • the primer of SEQ ID NO: 5 (NTM-1), primer of SEQ ID NO: 6 (NTM-1) and primer of SEQ ID NO: 7 (NTM-1) are 16S rRNA gene specific reverse primers.
  • a primer of SEQ ID NO: 8 (NTM-2) is a 16S rRNA gene specific reverse primer of non-acidic Mycobacterium tuberculosis.
  • the reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected.
  • nucleotide sequence 8 (5′-catcccacaccgctaccw-3 ′), it means a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11).
  • the primer of SEQ ID NO: 8 may be a primer set including about 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ at about 1: 1.
  • the 5 ′ end of the tuberculosis bacteria IS6110 gene detection probe and the anti-acidic tuberculosis 16S rRNA gene detection probe is FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS Labeled with one fluorescent labeling factor selected from the group consisting of RED, RED670 and NED, and with one fluorescent inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ at the 3 ′ end.
  • the label is characterized in that, the 5 'end of the probe for detecting the IS6110 gene of the Mycobacterium tuberculosis and 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors.
  • the 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe is labeled with VIC, and the 3' end is labeled with MGBNFQ
  • the 5 'end of the FDR 16S rRNA gene detection probe is labeled with FAM, and the 3' end is labeled with MGBNFQ.
  • the reverse primer of the nucleotide sequence 5 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1. Accordingly, the primers 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ of base sequence 5 may have a ratio of 2: 1: 1.
  • the reverse primer of the base sequence 6 and the reverse primer of the nucleotide sequence 8 may be 1: 1
  • the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10
  • Reverse primer of SEQ ID NO: 11 may be included 1: 1.
  • the reverse primer of the nucleotide sequence 7 and the reverse primer of the nucleotide sequence 8 may be 1: 1
  • the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10
  • Reverse primer of SEQ ID NO: 11 may be included 1: 1.
  • the present invention comprises the steps of: separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; One or more reverse primers (NTM-1) selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7, and a reverse primer of nucleotide sequence 8 (NTM- A primer set specific for the 16S rRNA gene of anti-acidic Mycobacterium tuberculosis comprising 2); And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-tuberculosis bacterium 16S rRNA gene of S
  • a forward primer of SEQ ID NO: 22 One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And it provides a primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
  • the forward primer of SEQ ID NO: 22 is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis.
  • the primer of SEQ ID NO: 5, primer of SEQ ID NO: 6, and primer of SEQ ID NO: 7 are 16S rRNA gene specific reverse primer (NTM-1).
  • a primer of SEQ ID NO: 8 (NTM-2) is a 16S rRNA gene specific reverse primer of non-acidic Mycobacterium tuberculosis.
  • the reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected.
  • the present invention provides a probe for detecting the acidic non-tuberculosis 16S rRNA gene of SEQ ID NO: 23.
  • the 5 ′ end of the probe is one fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED.
  • Labeled with a factor, and the 3 'end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3.
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 23 is one or more reverse primers selected from the group consisting of a forward primer of SEQ ID NO: 22, a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7 And it is a probe specific to the reaction product of the polymerase chain reaction method using a primer specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
  • the present invention provides a primer set specific to the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20;
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 23 and the Mycobacterium tuberculosis IS6110 gene detection probe of SEQ ID NO: 21 may be labeled with different detectable means.
  • the detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
  • the Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20.
  • the primer of the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all kinds of Mycobacterium tuberculosis (MTC).
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 23 may be a Taqman probe.
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20 Probe.
  • the primer of SEQ ID NO: 5, primer of SEQ ID NO: 6, and primer of SEQ ID NO: 7 are 16S rRNA gene specific reverse primer (NTM-1).
  • a primer of SEQ ID NO: 8 (NTM-2) is a 16S rRNA gene specific reverse primer of non-acidic Mycobacterium tuberculosis.
  • the reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected.
  • nucleotide sequence 8 (5′-catcccacaccgctaccw-3 ′), it means a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11).
  • the primer of SEQ ID NO: 8 may be a primer set including about 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ at about 1: 1.
  • the 5 'end of the tuberculosis bacteria IS6110 gene detection probe and the anti-acidic tuberculosis bacterium 16S rRNA gene detection probe is FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS Labeled with one fluorescent labeling agent selected from the group consisting of RED, RED670, and NED, and labeled with one fluorescent inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3 at the 3 ′ end.
  • the 5 'end of the probe for detecting the IS6110 gene of the Mycobacterium tuberculosis bacterium and the 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors.
  • the 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe is labeled with HEX and the 3' end is BHQ-1
  • the 5 'end of the FSA 16S rRNA gene detection probe is FAM and the 3' end is BHQ. -1 may be displayed.
  • the reverse primer of the nucleotide sequence 5 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1. Accordingly, the primers 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ of base sequence 5 may have a ratio of 2: 1: 1.
  • the reverse primer of the base sequence 6 and the reverse primer of the nucleotide sequence 8 may be 1: 1
  • the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10
  • Reverse primer of SEQ ID NO: 11 may be included 1: 1.
  • the reverse primer of the nucleotide sequence 7 and the reverse primer of the nucleotide sequence 8 may be 1: 1
  • the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10
  • Reverse primer of SEQ ID NO: 11 may be included 1: 1.
  • the step of separating the DNA from the sample A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 23; And it provides a method for detecting Mycobacterium tuberculo
  • the present invention provides a probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26.
  • the 5 'terminal of the probe is one of the fluorescence selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED It is labeled with a labeling factor, and the 3 'end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  • the probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26 is a polymerase chain using a common primer for amplifying 16S rRNA genes of Mycobacterium tuberculosis and anti-acidic tuberculosis comprising a forward primer set of SEQ ID NO: 24 and a reverse primer set of 25 It is a probe specific for the reaction product of Mycobacterium tuberculosis among the reaction products of the reaction method.
  • a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 28.
  • the probe for detecting an acidic non-tuberculosis 16S rRNA gene comprising the probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 28 is a 16S rRNA of Mycobacterium tuberculosis and antiacidic Mycobacterium tuberculosis comprising a forward primer set of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25 It is a probe specific to the reaction product of non-acidic tuberculosis bacteria among the reaction products of the polymerase chain reaction method using a common primer for amplifying a gene.
  • the 5 'terminal of the probe is one of the fluorescence selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED It is labeled with a labeling factor, and the 3 'end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  • the present invention provides a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and anti-acidic tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic tuberculosis 16S rRNA gene comprising a probe of the base sequence 27 and the probe of the base sequence 28.
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • Probe for detection of Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26 and probe for detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising the probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 28 may be labeled with different detectable means.
  • the detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto.
  • the excitation and emission wavelengths are different depending on the type, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
  • the forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 25 are primers capable of amplifying the 16S rRNA gene region of mycobacteria, and are a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis.
  • the forward primer of SEQ ID NO: 24 has a primer set including 5′-ggataagcctgggaaactgg-3 ′ (base sequence 29) and 5′-ggataagcttgggaaactgg-3 ′ (base sequence 30) in a ratio of about 1: 1 Can be.
  • the reverse primer of SEQ ID NO: 25 may be a primer set containing 5′-accccaccaacaagctgata-3 ′ (base sequence 31) and 5′-accccaccaactagctgata-3 ′ (base sequence 32) in a ratio of about 1: 1.
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26, the probe of SEQ ID NO: 27, and the probe of SEQ ID NO: 28 may be a Taqman probe.
  • the non-acidic non-tuberculosis bacterium 16S rRNA gene detection probe comprising a probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO. It was designed to target specifically distinguishable 16S rRNA bases.
  • the NTM-2 probe, the probe of SEQ ID NO: 28 is 5′-FAM-tggaaagcgtttggtagc-MGB-3 ′ (base sequence 33) and 5′-FAM-tggaaagtgtttggtagc-MGB-3 ′ (SEQ ID NO: 34) This may include about 1: 1.
  • the 5 ′ terminal of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising the probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26, the probe of SEQ ID NO: 27, and the probe of SEQ ID NO: 28
  • This is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED
  • the 3 'end is 6-TAMRA, BHQ Labeled with one fluorescence inhibitor selected from the group consisting of -1,2,3 and MGBNFQ, and a probe for detecting 16S rRNA gene of Mycobacterium tuberculosis bacterium of SEQ ID NO: 26 and a probe and base of SEQ ID NO: 27;
  • the 5 'end of the Mycobacterium tuberculosis 16S rRNA gene detection probe is labeled with VIC, and the 3' end is labeled with MGBNFQ, and the 5 'end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe is labeled with FAM and the 3' end is MGBNFQ. Can be displayed.
  • the step of separating the DNA from the sample A common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
  • the present invention provides a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of nucleotide sequence 37, a probe of nucleotide sequence 38 and a probe of nucleotide sequence 39.
  • the 5 'terminal of the probe is one of the fluorescence selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED Labeled with a marker factor, the 3 ′ end may be labeled with one fluorescence inhibitor (Quencher) selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ (molecular grove binding non-fluorescence quencher) have.
  • Quencher fluorescence inhibitor
  • Probe for detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a sequence of SEQ ID NO: 39 is a term comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36 It is a probe specific to the reaction product of the polymerase chain reaction method using a primer set specific for 16S rRNA gene of acidic mycobacterium tuberculosis.
  • the present invention provides a primer set specific to the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And it provides a tuberculosis bacteria and anti-acidic tuberculosis bacteria detection kit comprising a probe for detecting the non-acidic tuberculosis 16S rRNA gene selected from the group consisting of a probe of the nucleotide sequence 37, the probe of the nucleotide sequence 38 and the nucleotide sequence 39.
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • Probe of SEQ ID NO: 37 The probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of the probe of SEQ ID NO: 38 and the probe of SEQ ID NO: 39 and the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 are different detectable means.
  • the detectable means means a compound, a biomolecule, or a biomolecule mimetic that can be linked, coupled, or attached to a probe to confirm the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto.
  • the excitation and emission wavelengths are different depending on the type of fluorescent labeling factor, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used to determine whether they are detectable separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
  • the Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20.
  • the primer set of the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes ( MTCs ).
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA selected from the group consisting of the probe for detecting the Mycobacterium tuberculosis IS6110 gene of the nucleotide sequence 21 and the probe nucleotide sequence of the nucleotide sequence 37, the 38 probe, and the probe of the nucleotide sequence 39
  • the gene detection probe may be a Taqman probe.
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20 Probe.
  • Probe for detection of non-acidic mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of nucleotide sequence 37, a probe of nucleotide sequence 38 and a nucleotide sequence 39 comprises a forward primer of nucleotide sequence 35 and a reverse primer of nucleotide sequence 36
  • the forward primer of SEQ ID NO: 35 is an anti-acidic Mycobacterium tuberculosis 16S rRNA gene specific forward primer.
  • the forward primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected.
  • the 5 'end of the tuberculosis bacteria IS6110 gene detection probe and the anti-acidic tuberculosis bacterium 16S rRNA gene detection probe is FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS Labeled with one fluorescent labeling agent selected from the group consisting of RED, RED670, and NED, and the 3 ′ end is labeled with one fluorescent inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3
  • the 5 ′ end of the probe for detecting the IS6110 gene of the Mycobacterium tuberculosis and the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors.
  • the 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe is labeled with HEX and the 3' end is BHQ-1
  • the 5 'end of the FSA 16S rRNA gene detection probe is FAM and the 3' end is BHQ. -1 may be displayed.
  • the 16S rRNA gene detection probe of SEQ ID NO: 38 in the Mycobacterium tuberculosis and anti-acidic Mycobacterium tuberculosis detection kit is FAM-cctgagagggtgaccgg-BHQ1 (SEQ ID NO: 56) and FAM-cctgagagggtgtccgg-BHQ1 (SEQ ID NO: 57) is about 1 Can be included as: 1.
  • the step of separating the DNA from the sample A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculos
  • a base primer of SEQ ID NO: 61 provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer comprising a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 8.
  • the forward primer of SEQ ID NO: 61 is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis.
  • the primer of SEQ ID NO: 5 (NTM-1) and the primer of SEQ ID NO: 8 (NTM-2) are 16S rRNA gene specific reverse primers.
  • the reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected.
  • a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11) It may be a primer set including about 10 and the base sequence 11 in about 1: 1.
  • the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59;
  • a probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60;
  • a primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8;
  • it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63.
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • Gene detection probes may be labeled by different detectable means from each other.
  • the Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59.
  • the primer set specific for the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes (MTCs).
  • the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 60 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59 Probe.
  • the probe for detecting Mycobacterium tuberculosis IS6110 gene selected from the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 60 may be a Taqman probe.
  • the forward primer of SEQ ID NO: 61 is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis.
  • the primer of SEQ ID NO: 5 (NTM-1) and the primer of SEQ ID NO: 8 (NTM-2) are 16S rRNA gene specific reverse primers of non-acidic Mycobacterium tuberculosis.
  • the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63 is a primer specific for 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer comprising SEQ ID NO: 61, a primer of SEQ ID NO: 5, and a primer of SEQ ID NO: 8 A probe specific to the reaction product of polymerase chain reaction using a set.
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63 may be a Taqman probe.
  • the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Is labeled with one fluorescent labeling factor, and the 3 'end is labeled with one fluorescent inhibitor which is selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  • the 5 ′ end of the detection probe is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3 ′
  • the terminal is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ, and the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene are detected.
  • the 5 ′ end of the probe for labeled with different fluorescent labeling factors Can.
  • the step of separating the DNA from the sample A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 62 or a probe of SEQ ID NO: 63; And it provides a method for detecting the Mycobacterium tuber
  • a forward primer comprising a primer of nucleotide sequence 65, a primer of nucleotide sequence 66 and a primer of nucleotide sequence 67; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of nucleotide sequence 36.
  • the forward primer comprising the primer of SEQ ID NO: 65 (NTM-1), the primer of SEQ ID NO: 66 (NTM-2) and the primer of SEQ ID NO: 67 (NTM-3) is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis. to be.
  • the 16S rRNA gene forward primer of the anti-acidic tuberculosis bacterium was designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected.
  • nucleotide sequence 65 (′-tktggtggaaagctttgc-3 ′)
  • it is a primer set including 5′-tgtggtggaaagcttttgc-3 ′ (base sequence 69) and 5′-tttggtggaaagcttttgc-3 ′ (base sequence 70). It may be a primer set comprising about 1: 1 and 69 and nucleotide sequence 70.
  • a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 20;
  • a primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67;
  • it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the probe of SEQ ID NO: 39 or the base sequence 68.
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • Gene detection probes may be labeled by different detectable means from each other.
  • the Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20.
  • the primer set specific for the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes (MTCs).
  • the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 64 is specific for the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20 Probe.
  • the probe for detecting Mycobacterium tuberculosis IS6110 gene selected from the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 64 may be a Taqman probe.
  • the forward primer comprising the primer of SEQ ID NO: 65, the primer of SEQ ID NO: 66, and the primer of SEQ ID NO: 67 is a 16S rRNA gene specific forward primer of anti-acidic Mycobacterium tuberculosis.
  • the reverse primer of SEQ ID NO: 36 is a 16S rRNA gene specific reverse primer of non-acidic mycobacterium tuberculosis.
  • the probe of SEQ ID NO: 39 or the probe of SEQ ID NO: 68 is a 16S antifungal tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; and a reverse primer of SEQ ID NO: 36 Probe specific to the reaction product of the polymerase chain reaction method using a primer set specific to the rRNA gene.
  • the probe for detecting 16S rRNA gene of non-acidic Mycobacterium tuberculosis selected from the probe of SEQ ID NO: 39 or the probe of SEQ ID NO: 68 may be a Taqman probe.
  • the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Is labeled with one fluorescent labeling factor, and the 3 'end is labeled with one fluorescent inhibitor which is selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  • the 5 ′ end of the detection probe is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3 ′
  • the terminal is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ, and the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene are detected.
  • the 5 ′ end of the probe for labeled with different fluorescent labeling factors Can.
  • the step of separating the DNA from the sample A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 39 or a probe of SEQ ID NO: 68; And it provides a method for detecting Mycobacterium tuberculos
  • a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76.
  • the detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction.
  • Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
  • the anti-acidic Mycobacterium tuberculosis 16S rRNA comprising the probe for detecting the Mycobacterium tuberculosis IS6110 gene of base sequence 60 and the probe of base sequence 27 (NTM-1) and the probe of base sequence 76 (NTM-2) Gene detection probes may be labeled by different detectable means from each other.
  • the Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59.
  • the primer set specific for the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes (MTCs).
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59 Probe.
  • the probe for detecting the Mycobacterium tuberculosis IS6110 gene of nucleotide sequence 60 may be a Taqman probe.
  • the Mycobacterium tuberculosis and anti-acidic Mycobacterium tuberculosis detection kit includes a primer set specific for the 16S rRNA gene of Mycobacteria including a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75.
  • the forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 75 are primers capable of amplifying the 16S rRNA gene region of mycobacteria, and are a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis.
  • the forward primer of SEQ ID NO: 24 is a 16S rRNA gene specific forward primer of mycobacteria.
  • the primer set includes 5′-ggataagcctgggaaactgg-3 ′ (base sequence 29) and 5′-ggataagcttgggaaactgg-3 ′ (base sequence 30). It may be a primer set including 29 and nucleotide sequence 30 in about 1: 1.
  • the reverse primer of SEQ ID NO: 36 is a 16S rRNA gene specific reverse primer of mycobacteria.
  • the probe for detecting an acidic non-TB tuberculosis 16S rRNA gene comprising the probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 76 is specific for the 16S rRNA gene of Mycobacteria including a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75 Among the reaction products of the polymerase chain reaction method using a primer set, it is a probe specific to the reaction product of the polymerase chain reaction method of non-acidic tuberculosis bacteria.
  • the anti-acidic tuberculosis 16S rRNA gene detection probe including the probe of SEQ ID NO: 27 and the probe of base sequence 76 was designed to detect all kinds of anti-acidic tuberculosis bacteria to be detected.
  • the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Is labeled with one fluorescent labeling factor, and the 3 'end is labeled with one fluorescent inhibitor which is selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  • the 5 ′ end of the detection probe is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3 ′
  • the terminal is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ, and the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene are detected.
  • the 5 ′ end of the probe for labeled with different fluorescent labeling factors Can.
  • the step of separating the DNA from the sample A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
  • M. abscessus (AJ419970.1, AJ416940.1, AJ536038), M. acapulcensis (AF480575.1), M. africanum (AF480605.1), M. agri (AJ429045) .1), M. aichiense (X55598.1), M. alvei (NR_024859.1), M. asiaticum (X55604.1), M. aurum (FJ172298.1), M. austroafricanum (GU121552.1), M avium (NR_025584.1, AJ536037.1, EF521892.1), M. bohemicum (NR_026054.1), M. botniense (NR_028878.1), M.
  • duvalii (NR_026073.1), M. engbaekii (AF480577.1), M. fallax (AF480600.1) , M. farcinogenes (X55592.1), M. flavescens (AY734993.1), M. fortuitum (AY457066.1, AF480580.1, GU142933.1), M. gadium (NR — 026087.1), M. gastri (GU142918.1), M. genavense (NR_029223.1), M. gilvum (AB491971.1), M. goodii (AY457079.1), M. gordonae (GU142923.1), M. haemophilum (V06638.1) ), M.
  • NR_026011.1 M. heidelbergense (NR_025268.1), M. hiberniae (NR_026092.1), M. hodleri (NR_026286.1), M. immunogen (AJ011771.1), M. interjectum (X70961.1), M. intermedium (X67847.1), M. intracellulare (AY652958.1, AJ536036.1, X52927.1, M61684.1), M.kansasii (M29575.1, X15916.1), M lentiflavum (AF480583.1), M. mageritense (AY457076.1), M. malmoense (GQ153278.1), M.
  • marinum (AF456238.1, AY513243.1), M. microti (NR_025234.1), M. monacense (GU142931.1), M. moriokaense (AY859686.1), M. mucogenicum (AF480585.1), M. neoaurum (FJ172306.1), M. nonchromogenicum (DQ058406.1), M. obuense (X55597.1 ), M. paraffinicum (GQ153282.1), M. parafortuitum (NR_026285.1), M. peregrinum (AY457069.1) , M. phlei (AF480603.1), M. porcinum (AY457077.1), M.
  • thermoresistibile (GU142928.1), M. tilburgii (AJ580826.1), M. triplex (GQ153279 .1), M. triviale (DQ058405.1), M. tuberculosis (GU142936.1, GU142935.1, AY53603.1, X55588.1, X52917.1), M. tusciae (NR_024903.1), M. ulcerans
  • the base sequence data of 16S ribosomal RNA genes of (Z13990.1), M. vaccae (X55601.1) , M. wolinskyi (AY457083.1) and M. xenopi (X52929.1) were used for the analysis. Sequence data of the 16S ribosomal RNA gene of the mycobacteria was obtained from databases of the National Center for Biotechnology Information (NCBI).
  • the nucleotide sequence data of the 16S rRNA gene of the Mycobacteria strain was analyzed by Sequencher 4.9 to find a specific sequence region. In other words, the nucleotide sequence specific to Mycobacterium tuberculosis and the native nucleotide sequence of mycobacterium tuberculosis were not found.
  • Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe with 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
  • MTC Mycobacterium tuberculosis
  • Target Gene 16S rRNA
  • NTM-1 5′-cccacaccgcaaaagctt-3 ′ (base 5) or 5′-cccacaccgcaaaagct-3 ′ (base 6) or 5′-tcccacaccgcaaaagct-3 ′ (base 7)
  • NTM-2 5′-catcccacaccgctaccw-3 ′ (SEQ ID NO: 8)
  • strains of Mycobacterium tuberculosis 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated.
  • the ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
  • strains of Mycobacterium tuberculosis 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated.
  • the ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were carried out using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 66 ° C. for about 15 seconds. At this time, the composition of the reactants performing the double real-time polymerase chain reaction is shown in Table 4.
  • the forward primer and the reverse primer contained the same amount (10 pmole / ⁇ l) and the probe was 4 pmole / ⁇ l. Therefore, 1.25 ⁇ l of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole.
  • the total volume of 25uL of polymerase chain reaction was 25 ⁇ L.
  • the primer concentration was 0.5uM (12.5pmoles / 25 ⁇ l) and the probe 0.2uM (5 pmole/25 ⁇ l).
  • the concentration and volume of forward and reverse primers, probes of NTM were used the same as MTC.
  • NTM-1 reverse primer the primer of nucleotide sequence 5 was used as the NTM-2 reverse primer, and the primer of the nucleotide sequence 8 was used in the same amount.
  • NTM-2 reverse primers were designed such that 5′-catcccacaccgctacct-3 ′ (SEQ ID NO: 10) and 5′-catcccacaccgctacca-3 ′ (SEQ ID NO: 11) are present in equal amounts.
  • NTM forward primers are 5′-ggtaatctgccctgcac-3 ′ (SEQ ID NO: 12), 5′-ggtaacctgccctgcac-3 ′ (SEQ ID NO: 13), 5′-ggcaatctgccctgcac-3 ′ (SEQ ID NO: 14), 5′-ggcaacctgccctgcac-3 ′ (Base 15), 5′-ggtgatctgccctgcac-3 ′ (base 16), 5′-ggtgacctgccctgcac-3 ′ (base 17), 5′-ggcgatctgccctgcac-3 ′ (base 18) and 5′-ggcgacctgccctgcac- A primer set including 3 ′ (base sequence 19), wherein the primers of nucleotide sequence 12, nucleotide sequence 13, nucleotide sequence 14, nucleotide sequence 15, nucleotide sequence 16, nucleo
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). .
  • FRET Fluorescence Resonance Energy Transfer
  • 1 to 6 show the results obtained in the double real time polymerase chain reaction of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 1 and 2 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in the green and yellow channels of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 3 and 4 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
  • 5 and 6 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
  • Mycobacterium tuberculosis is the IS6110 gene in the yellow channel
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • + acidic non-tuberculosis NTM
  • the results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel.
  • the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
  • Mycobacterium tuberculosis complex (MTC: M. tuberculosis , M. bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe for detection of 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
  • MTC Mycobacterium tuberculosis
  • Target Gene 16S rRNA
  • NTM-1 5′-cccacaccgcaaaagctt-3 ′ (base 5) or 5′-cccacaccgcaaaagct-3 ′ (base 6) or 5′-tcccacaccgcaaaagct-3 ′ (base 7)
  • NTM-2 5′-catcccacaccgctaccw-3 ′ (SEQ ID NO: 8)
  • strains of Mycobacterium tuberculosis 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated.
  • the ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
  • strains of Mycobacterium tuberculosis 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated.
  • the ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 10 seconds, and annealing and extension processes at about 65 ° C. for about 15 seconds. At this time, the composition of the reactants to perform the double real-time polymerase chain reaction is shown in Table 2.
  • the forward primer and the reverse primer were contained in the same amount (10 pmole / ⁇ l) and the probe had 4 pmole / ⁇ l. Therefore, 1.25 ⁇ l of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole.
  • the total volume of the reactants for 25uL polymerization reaction was 25 ⁇ l, so the concentration of primer was 0.5uM (12.5pmoles / 25 ⁇ l) and the probe 0.2uM (5 pmole/25 ⁇ l).
  • the concentration and volume of forward and reverse primers, probes of NTM were used the same as MTC.
  • NTM-1 reverse primer the primer of base sequence 5 was used, and as the NTM-2 reverse primer, the primer of base sequence 8 was used in the same amount.
  • NTM-2 reverse primers were designed such that 5′-catcccacaccgctacct-3 ′ (SEQ ID NO: 10) and 5′-catcccacaccgctacca-3 ′ (SEQ ID NO: 11) are present in equal amounts.
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). .
  • FRET Fluorescence Resonance Energy Transfer
  • 7 to 12 show the results obtained in the double real-time polymerase chain reaction method of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 7 and 8 are graphs showing the change in the fluorescence intensity of the cycle number of the polymerase chain reaction in the yellow channel and the green channel of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 9 and 10 are graphs showing the change in fluorescence intensity for the number of cycles of the polymerase chain reaction in the yellow channel and the green channel of the double-real-time polymerase chain reaction of NRT.
  • 11 and 12 show y-axis for the number of cycles of polymerase chain reaction in the yellow channel and the green channel of the double real-time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • Mycobacterium tuberculosis is an IS6110 gene in the yellow channel
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM + acidic non-tuberculosis (NTM) is a yellow channel
  • the results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel.
  • the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
  • Example 3 Separation and Detection Method of Mycobacterium Tuberculosis and Anti-acidic Tuberculosis
  • Amplification of the 16S rRNA gene region of mycobacteria with consensus primers can be used to specifically distinguish Mycobacterium tuberculosis (MTC: M. tuberculosis , M. bovis , M. africanum , M. microti ) from acid-free tuberculosis (NTM) 16S rRNA base site was used as a Taqman probe.
  • MTC Mycobacterium tuberculosis
  • M. bovis M. bovis
  • M. africanum M. microti
  • NTM acid-free tuberculosis
  • MTC Mycobacterium tuberculosis
  • NTM-1 5′-FAM-tggtggaaagcttttgc-MGB-3 ′ (SEQ ID NO: 27)
  • NTM-2 5′-FAM-tggaaagygtttggtagc-MGB-3 ′ (SEQ ID NO: 28)
  • strains of Mycobacterium tuberculosis 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated.
  • the ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
  • strains of Mycobacterium tuberculosis 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated.
  • the ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were carried out using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 66 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 3.
  • the forward primer and the reverse primer of the common primer of the mycobacteria were contained in the same amount (10 pmole / ⁇ l), and the MTC probe, NTM-1 probe and NTM-2 probe each contained 4 pmole / ⁇ l.
  • 1.25 ⁇ l of primer-probes mix used for the reaction had forward and reverse primers of 12.5 pmole of common primer and 5 pmole of MTC, NTM-1, and NTM-2 probes, respectively.
  • the total volume of the reaction product for performing the polymerization reaction of 25 ⁇ l was 25 ⁇ l.
  • the concentration of the primer was 0.5 ⁇ M (12.5 pmole / 25 ⁇ l), and the probe of MTC, NTM-1, NTM-2 was 0.2 ⁇ M ( 5 pmole / 25 ⁇ l).
  • the forward primers of the nucleotide sequence 24 of the mycobacteria were designed such that 5'-ggataagcctgggaaactgg-3 '(base sequence 29) and 5'-ggataagcttgggaaactgg-3' (base sequence 30) were present in the same amount of 6.25 pmole.
  • the reverse primer of SEQ ID NO: 25 was designed such that 5'-accccaccaacaagctgata-3 '(base sequence 31) and 5'-accccaccaactagctgata-3' (base sequence 32) were present in equal amounts of 6.25 pmole.
  • the probe of SEQ ID NO: 28, which is an NTM-2 probe has the same amount of 5'-FAM-tggaaagcgtttggtagc-MGB-3 '(base sequence 33) and 5'-FAM-tggaaagtgttggtagc-MGB-3' (base sequence 34). It is designed to exist by pmole.
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). .
  • FRET Fluorescence Resonance Energy Transfer
  • 13 to 18 show the results obtained in the double real time polymerase chain reaction method of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 13 and 14 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 15 and 16 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
  • 17 and 18 are y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (MTC) + acidic non-TB tuberculosis (NTM), respectively, indicating a change in fluorescence intensity. It is a graph.
  • Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe with 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
  • MTC Mycobacterium tuberculosis
  • Target Gene 16S rRNA
  • FAM-tagccggcctgagagggtg-BHQ1 base sequence 37 or FAM-cctgagagggtgwccggcc-BHQ1 (base sequence 38) or FAM-cgggtagccggcctgagag-BHQ1 (base sequence 39)
  • gordonae KCTC 9513 M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950 , M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571 , M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M.
  • neoaurum KCTC 19096 M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979 , M. peregrinum KCTC 9615, KMRC 75002, M. phlei KCTC 9689 , M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108 , M.
  • szulgai KCTC 9520 KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M vanbaalenii KCTC 9966 , M. wolinskyi ATCC 700010, M. xenopi KMRC 42001.
  • M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens.
  • ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 12 seconds, and annealing and extension process at about 63 ° C. for about 12 seconds. At this time, the composition of the reactants performing the double real-time polymerase chain reaction is shown in Table 4.
  • the forward primer and the reverse primer contained the same amount (10 pmole / ⁇ l) and the probe was 4 pmole / ⁇ l. Therefore, 1.25 ⁇ l of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole.
  • the total volume of the reaction product to perform 25 ⁇ L of polymerization chain reaction was 25 ⁇ L.
  • the concentration of the primer was 0.5 ⁇ M (12.5 pmoles / 25 ⁇ L) and the probe was 0.2 ⁇ M (5 pmole / 25 ⁇ L).
  • the concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
  • NTM forward primer (base sequence 35) is 5′-catgtcttgtgggggaaagctt-3 ′ (base sequence 40), 5′-catgttttgtgggggaaagctt-3 ′ (base sequence 41), 5′-catgtcttctgggggaaagctt-3 ′ (base sequence 42) , 5′-catgtcttgtggtggaaagctt-3 ′ (base 43), 5′-catgtcttgtggggcaaagctt-3 ′ (base 44), 5′-catgttttctgggggaaagctt-3 ′ (base 45), 5′-catgtcttctggtggaaagctt-3 ′ (base sequence) 46), 5′-catgtcttggtggaaagctt-3 ′ (base sequence 47), 5′-catgttttggggcaaag
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). .
  • FRET Fluorescence Resonance Energy Transfer
  • the probe of SEQ ID NO: 38 is designed so that FAM-cctgagagggtgaccggcc-BHQ1 (base sequence 56) and FAM-cctgagagggtgtccggcc-BHQ1 (base sequence 57) are present in equal amounts.
  • 19 to 24 show the results obtained in the double real-time polymerase chain reaction method of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 19 and 20 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 21 and 22 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
  • FIG. 23 and FIG. 24 show y-axis for the number of cycles of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • the tuberculosis bacterium is an IS6110 gene in the yellow channel
  • the acidic non-tuberculosis bacterium is the 16S rRNA gene in the green channel
  • Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is the yellow channel
  • the results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel.
  • the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
  • Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe for detection of 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
  • MTC Mycobacterium tuberculosis
  • Target Gene 16S rRNA
  • Example 5-1 Dual real-time polymerase chain reaction using Taqman probe of SEQ ID NO: 21 as a probe for detecting Mycobacterium tuberculosis (MTC) and Taqman probe of SEQ ID NO: 62 as a probe for detecting non-acidic Mycobacterium tuberculosis>
  • gordonae KCTC 9513 M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950 , M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571 , M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M.
  • neoaurum KCTC 19096 M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979 , M. peregrinum KCTC 9615, KMRC 75002, M. phlei KCTC 9689 , M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108 , M.
  • szulgai KCTC 9520 KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M vanbaalenii KCTC 9966 , M. wolinskyi ATCC 700010, M. xenopi KMRC 42001.
  • M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens.
  • ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 65 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 5.
  • the forward primer and the reverse primer contained the same amount (10 pmole / ⁇ l) and the probe was 4 pmole / ⁇ l. Therefore, 1.25 ⁇ l of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole.
  • the total volume of 25uL of polymerase chain reaction was 25 ⁇ L.
  • the primer concentration was 0.5uM (12.5pmoles / 25 ⁇ l) and the probe 0.2uM (5 pmole/25 ⁇ l).
  • the concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . It is designated to display in the green channel (510 ⁇ 5nm) when the FAM TM is developed on the real time monitor, and in the yellow channel (555 ⁇ 5nm) when the Hex TM or VIC TM is developed. Fluorescence was observed in the green channel and the yellow channel.
  • FRET Fluorescence Resonance Energy Transfer
  • Example 5-2 Dual real-time polymerase chain reaction using Taqman probe of SEQ ID NO: 21 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of SEQ ID NO: 63 as a probe for detecting acidic non-TB bacteria>
  • Example 5-1 Dual real-time in substantially the same manner as Example 5-1 and Taqman probe of SEQ ID NO: 21 as the probe for detecting Mycobacterium tuberculosis (MTC), and Taqman probe of SEQ ID NO: 63 as the probe for detecting the non-acidic Mycobacterium tuberculosis bacterium Polymerase chain reaction was performed.
  • Example 5-1 Dual real-time in the same manner as in Example 5-1 except that a Taqman probe of SEQ ID NO: 60 was used as a probe for detection of Mycobacterium tuberculosis (MTC), and a Taqman probe of SEQ ID NO: 62 was used as a probe for detecting an acidic non-TB bacterium. Polymerase chain reaction was performed.
  • MTC Mycobacterium tuberculosis
  • SEQ ID NO: 62 was used as a probe for detecting an acidic non-TB bacterium. Polymerase chain reaction was performed.
  • Example 5-4 Dual real-time polymerase chain reaction using Taqman probe of base sequence 60 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base sequence 63 as a probe for detecting non-malignant tuberculosis bacteria>
  • 25 to 30 show the results obtained in the double real-time polymerase chain reaction method of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 25 and 26 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • FIG. 27 and FIG. 28 are graphs showing changes in fluorescence intensity of the cycle number of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction of NRT.
  • 29 and 30 show y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • Mycobacterium tuberculosis is the IS6110 gene in the yellow channel
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • acidic non-tuberculosis NTM
  • NTM acidic non-tuberculosis
  • the results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel.
  • the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
  • Example 6 Separation and Detection Method of Mycobacterium Tuberculosis and Antiacidic Tuberculosis
  • Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe for detection of 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
  • MTC Mycobacterium tuberculosis complex
  • Target Gene 16S rRNA
  • NTM-1 5′-tktggtggaaagcttttgc-3 ′ (SEQ ID NO: 65)
  • NTM-2 5′-ggtgwgtggtgcaaagctt-3 ′ (SEQ ID NO: 66)
  • NTM-3 5′-tggtggaaagcgtttggt-3 ′ (SEQ ID NO: 67)
  • M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens.
  • ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 64 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 6.
  • the forward primer and the reverse primer contained the same amount (10 pmole / ⁇ l) and the probe was 4 pmole / ⁇ l. Therefore, 1.25 ⁇ l of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole.
  • the total volume of 25uL of polymerase chain reaction was 25 ⁇ L.
  • the primer concentration was 0.5uM (12.5pmoles / 25 ⁇ l) and the probe 0.2uM (5 pmole/25 ⁇ l).
  • the concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . It is designated to display in the green channel (510 ⁇ 5nm) when the FAM TM is developed on the real time monitor, and in the yellow channel (555 ⁇ 5nm) when the Hex TM or VIC TM is developed. Fluorescence was observed in the green channel and the yellow channel.
  • FRET Fluorescence Resonance Energy Transfer
  • Example 6-2 Dual real-time polymerase chain reaction using Taqman probe of SEQ ID NO: 21 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of SEQ ID NO: 68 as a probe for detecting acidic non-TB bacteria>
  • Example 6-1 Dual real-time in substantially the same manner as in Example 6-1 except that the Taqman probe of SEQ ID NO: 21 was used as a probe for the detection of Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 68 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
  • MTC Mycobacterium tuberculosis
  • SEQ ID NO: 68 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
  • Example 6-3 Dual real-time polymerase chain reaction using Taqman probe of base sequence 64 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base sequence 39 as a probe for detecting non-malignant tuberculosis bacteria>
  • Example 6-1 Dual real-time in substantially the same manner as in Example 6-1, except that the Taqman probe of SEQ ID NO: 64 was used as a probe for the detection of Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 39 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
  • MTC Mycobacterium tuberculosis
  • SEQ ID NO: 39 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
  • Example 6-4 Dual real-time polymerase chain reaction using Taqman probe of base sequence 64 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base 68 as a probe for detecting non-malignant tuberculosis bacteria>
  • Example 6-1 Dual real-time in the same manner as in Example 6-1 except that the Taqman probe of SEQ ID NO: 64 was used as a probe for the detection of Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 68 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
  • MTC Mycobacterium tuberculosis
  • SEQ ID NO: 68 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
  • 31 to 36 show the results obtained in the double real time polymerase chain reaction method of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 31 and 32 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 33 and 34 are graphs showing changes in fluorescence intensity of cycles of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction (NTM).
  • 35 and 36 show the y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
  • Mycobacterium tuberculosis is the IS6110 gene in the yellow channel
  • acidic non-tuberculosis NTM
  • Mycobacterium tuberculosis MTC
  • + acidic non-tuberculosis NTM
  • the results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel.
  • the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
  • Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB can specifically distinguish 16S rRNA genes.
  • 16S rRNA base site was used as a Taqman probe.
  • a primer capable of amplifying the 16S rRNA gene region of Mycobactera was used as a common primer. The primer for the detection site was designed using the Primer3 program.
  • MTC Mycobacterium tuberculosis complex
  • Target Gene 16S rRNA
  • NTM-1 5′-FAM-tggtggaaagcttttgc-MGB-3 ′ (SEQ ID NO: 27)
  • NTM-2 5′-FAM-ccacaccgctaccaaac-MGB-3 ′ (SEQ ID NO: 76)
  • M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens.
  • ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
  • the DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500 ⁇ l of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 ⁇ l of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • the DNA of mycobacteria grown in solid medium was extracted as follows. 500 ⁇ l of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
  • the sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded.
  • the supernatant was removed, and 100 ⁇ l of 5% chelex resin (Biorad, USA) and 1 ⁇ l of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
  • Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 65 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 7.
  • the forward primer and the reverse primer contained the same amount (10 pmole / ⁇ l) and the probe was 4 pmole / ⁇ l. Therefore, 1.25 ⁇ l of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole.
  • the total volume of 25uL of polymerase chain reaction was 25 ⁇ L.
  • the primer concentration was 0.5uM (12.5pmoles / 25 ⁇ l) and the probe 0.2uM (5 pmole/25 ⁇ l).
  • the concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
  • the dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). .
  • FRET Fluorescence Resonance Energy Transfer
  • 37 to 42 show the results obtained in the double real time polymerase chain reaction method of the strains.
  • the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F).
  • 37 and 38 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
  • 39 and 40 are graphs showing changes in fluorescence intensity of the cycle number of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction of NRT.
  • 41 and 42 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
  • the MTC is an IS6110 gene in the yellow channel
  • the nonacidic tuberculosis bacterium (NTM) is the 16S rRNA gene in the green channel
  • the tuberculosis bacterium (MTC) + the acidic non-tuberculosis bacterium (NTM) is the yellow channel, respectively.
  • the results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel.
  • the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
  • the tuberculosis specific base sequence and the native nucleotide sequence of the anti-acidic non-tuberculosis tuberculosis which is absent from the tuberculosis bacterium may be forward or reverse primers; And / or designed with a probe, a test kit was prepared, and the double- real-time polymerase chain reaction method was performed to evaluate the standard strains used to search for specific sequences of the tuberculosis bacteria and the non-acidic tuberculosis bacteria. It was confirmed that it is a means for evaluating anti-acidic tuberculosis bacteria. Therefore, it is possible to provide a means for effectively detecting various kinds of Mycobacterium tuberculosis bacteria and homeostatic non-tuberculosis bacteria.
  • the primers and / or probes for detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria that can detect gene sequences specific for Mycobacterium tuberculosis and anti-acidic tuberculosis bacteria have high diagnostic susceptibility and specificity to Mycobacterium tuberculosis and anti-acidic tuberculosis bacteria.
  • clinical diagnostic means capable of more efficiently detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria in the sample at the same time can be provided.
  • the present invention provides a primer set and / or probe for detecting a gene sequence specific to Mycobacterium tuberculosis and Mycobacterium tuberculosis, and a detection kit and a method for detecting tuberculosis and / or mycobacterium tuberculosis. It is possible to provide a means of detecting clinical diagnostics, which may be used in various industries such as hospitals and research institutes.
  • SEQ ID NO: 1 is a forward primer specific for the IS6110 gene of Mycobacterium tuberculosis complex.
  • SEQ ID NO: 2 is a reverse primer specific for the IS6110 gene of Mycobacterium tuberculosis complex.
  • SEQ ID NO: 3 is a probe specific for the IS6110 gene of Mycobacterium tuberculosis complex.
  • SEQ ID NO: 4 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 5 is a reverse primer (NTM-1) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 6 is a reverse primer (NTM-1) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 7 is a reverse primer (NTM-1) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 8 is a reverse primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 9 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 10 is a reverse primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 11 is a reverse primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 12 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 13 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 14 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 15 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 16 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 17 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 18 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 19 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 20 is a reverse primer specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
  • SEQ ID NO: 21 is a probe specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
  • SEQ ID NO: 22 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 23 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 24 is a forward universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 25 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 26 is a probe specific for the 16S rRNA gene of Mycobacterium tuberculosis complex.
  • SEQ ID NO: 27 is a probe (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 28 is a probe (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 29 is a forward universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 30 is a forward universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 31 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 32 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 33 is a probe (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 34 is a probe (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 35 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 36 is a reverse primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 37 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 38 is a probe specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 39 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 40 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 41 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 42 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 43 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 44 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 45 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 46 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 47 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 48 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 49 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 50 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 51 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 52 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 53 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 54 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 55 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 56 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 57 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 58 is a forward primer specific for the IS6110 gene of Mycobacterium tuberculosis complex.
  • SEQ ID NO: 59 is a reverse primer specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
  • SEQ ID NO: 60 is a probe specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
  • SEQ ID NO: 61 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 62 is a probe specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 63 is a probe specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 64 is a probe specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
  • SEQ ID NO: 65 is a forward primer (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 66 is a forward primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 67 is a forward primer (NTM-3) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 68 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 69 is a forward primer (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 70 is a forward primer (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 71 is a forward primer (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 72 is a forward primer (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 73 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 74 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
  • SEQ ID NO: 75 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
  • SEQ ID NO: 76 is a probe (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.

Abstract

The present invention provides: a primer set and/or a probe for detecting Mycobacterium tuberculosis and nontuberculous mycobacteria specific for a Mycobacterium tuberculosis-specific IS6110 gene or 16S rRNA gene and a nontuberculous mycobacteria-specific 16S rRNA gene; a kit for detecting Mycobacterium tuberculosis and nontuberculous mycobacteria, containing the same; and a method for detecting Mycobacterium tuberculosis and nontuberculous mycobacteria through dual real-time polymerase chain reaction by using the same. The present invention can provide a clinical diagnosis means capable of more effectively detecting and analyzing Mycobacterium tuberculosis and/or nontuberculous mycobacteria at the same time.

Description

이중 실시간 중합효소연쇄반응법을 이용한 결핵균과 항산성비결핵균의 검출 방법Detection of Mycobacterium Tuberculosis and Acidophilic Mycobacterium Tuberculosis Using Dual Real-time Polymerase Chain Reaction
본 발명은 결핵균과 항산성비결핵균의 검출 방법에 관한 것이다. 보다 상세하게는 결핵균과 항산성비결핵균에 특이적인 유전자 염기서열을 검출할 수 있는 결핵균과 항산성비결핵균 검출용 프라이머 세트 및/또는 프로브, 이를 포함하는 결핵균과 항산성비결핵균 검출 키트 및 이를 이용하는 이중 실시간 중합효소연쇄반응법을 이용한 결핵균과 항산성비결핵균의 동시 검출 방법에 관한 것이다.The present invention relates to a method for detecting Mycobacterium tuberculosis bacteria and anti-acidic non-tuberculosis bacteria. More specifically, the tube set and / or probes for detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria that can detect the gene sequence specific to the tuberculosis bacteria and acidic non-TB tuberculosis, tuberculosis bacteria and acidic non-TB tuberculosis detection kit and dual real-time polymerization using the same The present invention relates to a method for simultaneously detecting Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis using enzyme chain reaction.
항산성비결핵균(nontuberculous mycobacteria)은 토양이나 물 등 자연환경 등에 널리 존재하는 비병원성 균으로 인식되어 왔다. 그러나, 1980년대 후천성면역결핍증(AIDS)이 유행하면서, 상기 균이 후천성 면연결핍증 환자의 기회 균주로서 항산성비결핵균 유발 질환이 확인되고, 정상 환자에게서도 감염을 일으킬 수 있음이 알려지면서 임상적 중요성에 대한 인식이 확산되었다.Nontuberculous mycobacteria have been recognized as non-pathogenic bacteria widely present in natural environments such as soil and water. However, in the 1980s, AIDS has been prevalent, and it has been confirmed that the bacterium is an opportunistic strain of M. tuberculosis as an opportunistic strain of patients with AIDS, and that it can cause infection in normal patients. Awareness has spread.
최근에는 결핵균에 의한 유병률이 낮은 미국 및 유럽 여러 나라 항산성비결핵균에 의한 감염증이 증가하고 있고, 국내에서도 결핵 유병율이 저하하고 있으나 상대적으로 항산성비결핵균에 의한 감염이 증가하는 추세이다. 2009년 액체 결핵배양법이 보험급여 됨에 따라 액체결핵배양 검사를 실시하는 검사실이 늘고 있으며 액체배지를 사용하여 결핵을 배양하는 경우 기존의 고체배지를 사용한 결핵 배양보다 항산성 비결핵균이 더 검출되고 있다. 보고에 의하면 결핵도말 및 배양 양성 검체의 약 12%에서 항산성비결핵균이 분리되고 있으며, 일본, 홍콩, 우리나라의 경우 객담에서 분리된 항산성비결핵균의 약 10~20%가 폐질환을 야기하고 미국, 캐나다, 서유럽 등에서는 약 40~50%가 폐질환을 야기하는 것으로 알려지고 있다.Recently, infectious diseases caused by acid-resistant non-tuberculosis bacteria have been increasing in the United States and Europe, where the prevalence of tuberculosis bacteria is low, and the prevalence of tuberculosis in Korea is decreasing, but infection by anti-acidic tuberculosis bacteria is relatively increasing. As the Liquid Tuberculosis Culture Act was insured in 2009, an increasing number of laboratories conducting liquid tuberculosis culture examinations have been detected. When culturing tuberculosis using liquid media, more acid-resistant non-tuberculosis bacteria are detected than tuberculosis cultures using solid media. According to the report, about 12% of tuberculosis smears and culture-positive specimens are isolated from non-acidic non-tuberculosis bacteria, and in Japan, Hong Kong and Korea, about 10-20% of the anti-acidic tuberculosis bacteria isolated from sputum cause lung disease and In the United States, Canada and Western Europe, about 40-50% are known to cause lung disease.
그런데 항산성비결핵균에 의한 폐질환은 천천히 진행되는 폐결핵과 유사하여 오진되기 쉬우나, 결핵균과 항산성비결핵균에 감수성을 보이는 약제가 달라 치료 약제를 선택하기 위해 신속하고 정확한 결핵균과 항산성비결핵균의 분리검출 방법이 요청되고 있다.However, pulmonary disease caused by acidic non-tuberculosis bacillus is easy to be misdiagnosed because it is similar to the slowly progressing pulmonary tuberculosis. However, since the drugs showing susceptibility to tuberculosis bacteria and anti-acidic non-tuberculosis bacillus are different, the rapid and accurate method of separating and detecting tuberculosis bacteria and acidic non-tuberculosis bacillus Is being requested.
하지만, 현재 시판되고 있는 결핵균과 항산성비결핵균을 검출하는데 사용되는 검사시약은 항산성비결핵균에만 고유한 부위가 아니고, 결핵균도 가지고 있는 염기서열 부위를 사용함으로써, 특정 농도의 결핵균만 있을 시에 결핵균 검출 프라이머에는 반응하지 않고 항산성비결핵균의 프라이머에만 반응 하는 항산성비결핵균으로 잘못 동정되거나, 항산성비결핵균과 결핵균이 동시에 존재하는 경우 항산성비결핵균만 검출되는 경우도 관찰되는 등 검출 및 진단 정확성이 떨어지는 문제가 발생하고 있다. 따라서, 결핵균에는 없고 항산성비결핵균의 고유염기서열을 인식할 수 있는 프라이머 세트 및/또는 프로브 및 이를 이용한 신속하고 정확한 결핵균과 항산성비결핵균의 분리 검출 방법이 요청되고 있다.However, currently available test reagents for detecting tuberculosis bacteria and non-acidic non-tuberculosis bacteria are not unique to acidic non-tuberculosis bacteria. The problem of poor detection and diagnosis accuracy is that it is incorrectly identified as an acidic non-tuberculosis bacterium that reacts only to the primers of an acidic non-tuberculosis bacterium without reacting to the primer, or when only an acidic non-tuberculosis bacillus is detected when both the acidic non-tuberculosis bacillus and tuberculosis bacteria are present. It is happening. Accordingly, there is a need for a primer set and / or probe that is not present in the tuberculosis bacteria and capable of recognizing the native nucleotide sequence of the nonacidic tuberculosis bacteria, and a rapid and accurate method for separating and detecting the tuberculosis bacteria and the acidic non-tuberculosis bacteria.
본 발명의 목적은 결핵균과 항산성비결핵균의 정확한 검출과 진단을 위한 결핵균 특이적 IS6110 유전자에 높은 검출력을 가진 프라이머 세트와 항산성비결핵균의 16S rRNA 유전자에 높은 검출력을 가진 프라이머 세트를 제공하는 데 있다.SUMMARY OF THE INVENTION An object of the present invention is to provide a primer set having a high detection ability in Mycobacterium tuberculosis-specific IS6110 gene for accurate detection and diagnosis of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis, and a primer set having high detection ability in 16S rRNA gene of mycobacterium tuberculosis.
본 발명의 다른 목적은 결핵균과 항산성비결핵균의 정확한 검출과 진단을 위한 결핵균 특이적 IS6110 유전자 또는 16S rRNA 유전자에 높은 검출력을 가진 프로브와 항산성비결핵균의 16S rRNA 유전자에 높은 검출력을 가진 프로브를 제공하는 데 있다.It is another object of the present invention to provide a probe having high detection ability in Mycobacterium tuberculosis-specific IS6110 gene or 16S rRNA gene for accurate detection and diagnosis of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis and 16S rRNA gene of mycobacterium tuberculosis. There is.
본 발명의 또 다른 목적은 본 발명의 목적은 상기 프라이머 세트 및/또는 프로브를 포함하는 결핵균과 항산성비결핵균의 검출 키트를 제공하는데 있다.Another object of the present invention is to provide a detection kit of Mycobacterium tuberculosis and anti-acidic Mycobacterium tuberculosis comprising the primer set and / or probe.
본 발명의 또 다른 목적은 상기 프라이머 및/또는 프로브를 이용하는 이중 실시간 중합효소연쇄반응법(duplex real-time polymerase chain reaction)을 이용하여 결핵균과 항산성비결핵균을 정확하게 검출과 진단할 수 있는 방법을 제공하는 데 있다.It is another object of the present invention to provide a method for accurately detecting and diagnosing tuberculosis bacteria and non-acidic tuberculosis bacteria by using a duplex real-time polymerase chain reaction using the primers and / or probes. There is.
본 발명은 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다. The present invention provides a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 9.
본 발명은 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 4의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.The present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 1 and a reverse primer of nucleotide sequence 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And it provides a Mycobacterium tuberculosis bacillus and anti-acidic Mycobacterium tuberculosis detection kit comprising a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 4의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.The present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-tuberculosis bacterium 16S rRNA gene of SEQ ID NO: 9; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 염기서열 22의 정방향 프라이머; 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머; 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 제공한다.The present invention is a forward primer of SEQ ID NO: 22; One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And it provides a primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
본 발명은 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다. The present invention provides a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 23.
본 발명은 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다. The present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And it provides a tuberculosis bacteria and anti-acidic tuberculosis bacteria detection kit comprising a probe for detecting the non-acidic tuberculosis 16S rRNA gene of SEQ ID NO: 23.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머; 및 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다. The present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primers specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 23; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다. The present invention provides a probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26.
본 발명은 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다. The present invention provides a probe for detecting non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28.
본 발명은 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트; 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브; 및 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다. The present invention provides a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic tuberculosis 16S rRNA gene comprising a probe of the base sequence 27 and the probe of the base sequence 28.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트; 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브; 및 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다. The present invention comprises the steps of separating the DNA from the sample; A common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다.The present invention provides a probe for detecting non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39.
본 발명은 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출키트를 제공한다. The present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for detecting the anti-acidic tuberculosis 16S rRNA gene selected from the group consisting of a probe of the nucleotide sequence 37, the probe of the nucleotide sequence 38 and the nucleotide sequence 39.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다. The present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 염기서열 61의 정방향 프라이머; 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 제공한다. The present invention is a forward primer of SEQ ID NO: 61; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer comprising a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 8.
본 발명은 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다. The present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다. The present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 62 or a probe of SEQ ID NO: 63; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머; 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 제공한다. The present invention provides a primer comprising a primer of nucleotide sequence 65, a primer of nucleotide sequence 66 and a primer of nucleotide sequence 67; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of nucleotide sequence 36.
본 발명은 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다. The present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the probe of SEQ ID NO: 39 or the base sequence 68.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.The present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 39 or a probe of SEQ ID NO: 68; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.The present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76.
본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.The present invention comprises the steps of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
본 발명은 결핵균과 항산성비결핵균에 특이적인 유전자 염기서열을 검출할 수 있는 결핵균과 항산성비결핵균 검출용 프라이머 세트 및/또는 프로브, 및 이를 포함하는 검출키트 및 이를 이용하는 이중 실시간 중합효소연쇄반응법에 의한 결핵균과 항산성비결핵균의 검출 방법을 제공할 수 있다. 상기 방법에 의하면, 결핵균과 항산성비결핵균에 특이적인 프라이머 및/또는 프로브를 이용하여 결핵균 및/또는 항산성비결핵균을 동시에 보다 효율적으로 검출할 수 있는 임상진단 수단을 제공할 수 있다.The present invention provides a primer set and / or a probe for detecting Mycobacterium tuberculosis and Mycobacterium tuberculosis, which can detect gene sequences specific for Mycobacterium tuberculosis and Mycobacterium tuberculosis, and a detection kit comprising the same and a dual real-time polymerase chain reaction method using the same. It is possible to provide a method for detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria. According to the above method, it is possible to provide a clinical diagnostic means capable of more efficiently detecting Mycobacterium tuberculosis and / or Mycobacterium tuberculosis at the same time using primers and / or probes specific for Mycobacterium tuberculosis and Mycobacterium tuberculosis.
도 1 및 도 2는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.1 and 2 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in the green and yellow channels of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 3 및 도 4는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.3 and 4 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
도 5 및 도 6는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.5 and 6 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
도 7 및 도 8는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 노랑채널과 녹색채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.7 and 8 are graphs showing the change in the fluorescence intensity of the cycle number of the polymerase chain reaction in the yellow channel and the green channel of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 9 및 도 10는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 노랑채널과 녹색채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.9 and 10 are graphs showing the change in fluorescence intensity for the number of cycles of the polymerase chain reaction in the yellow channel and the green channel of the double-real-time polymerase chain reaction of NRT.
도 11 및 도 12는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 노랑채널과 녹색채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.11 and 12 show y-axis for the number of cycles of polymerase chain reaction in the yellow channel and the green channel of the double real-time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
도 13 및 도 14는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.13 and 14 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 15 및 도 16는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.15 and 16 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
도 17 및 도 18는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.17 and 18 are y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (MTC) + acidic non-TB tuberculosis (NTM), respectively, indicating a change in fluorescence intensity. It is a graph.
도 19 및 도 20는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.19 and 20 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 21 및 도 22는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.21 and 22 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM).
도 23 및 도 24는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.FIG. 23 and FIG. 24 show y-axis for the number of cycles of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
도 25 및 도 26는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.25 and 26 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 27 및 도 28는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.FIG. 27 and FIG. 28 are graphs showing changes in fluorescence intensity of the cycle number of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction of NRT.
도 29 및 도 30는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다..29 and 30 show y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It's a graph.
도 31 및 도 32는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.31 and 32 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 33 및 도 34는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.33 and 34 are graphs showing changes in fluorescence intensity of cycles of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction (NTM).
도 35 및 도 36는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.35 and 36 show the y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
도 37 및 도 38는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.37 and 38 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively.
도 39 및 도 40는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다 .39 and 40 are graphs showing changes in fluorescence intensity of the cycle number of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction (NTM).
도 41 및 도 42는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.41 and 42 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
상기 목적을 달성하기 위하여, 본 발명은 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다.In order to achieve the above object, the present invention provides a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9.
본 발명의 일실시예에 따르면 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ(molecular grove binding non-fluorescence quencher)로 이루어진 군으로부터 선택된 1종의 형광 억제 물질(Quencher)로 표지될 수 있다.According to an embodiment of the present invention, the 5 ′ end of the probe is one fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a factor, and the 3 ′ end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and molecular grove binding non-fluorescence quencher (MBGBFQ). .
상기 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 4의 정방향 프라이머; 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머; 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.Probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9 is a forward primer of SEQ ID NO: 4; One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And it is a probe specific to the reaction product of the polymerase chain reaction method using a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
상기 다른 목적을 달성하기 위하여, 본 발명은 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 4의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.In order to achieve the above another object, the present invention provides a primer set specific to the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 1 and a reverse primer of nucleotide sequence 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And it provides a Mycobacterium tuberculosis bacillus and anti-acidic Mycobacterium tuberculosis detection kit comprising a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
상기 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다. 상기 검출가능한 수단은 프로브에 연결, 결합, 또는 부착시켜 통상적인 방식으로 밀도, 농도, 양 등을 확인할 수 있는 화합물, 생체 분자 또는 생체 분자 모방체 등을 의미한다. 그 예로, 통상적으로 사용되는 형광 표지인자, 발광물질, 생발광물질, 동위원소 등이 있으나, 이로 한정되는 것은 아니다. 형광 표지 인자는 종류에 따라 여기 및 방사 파장이 다르면 사용방법 또한 상이하므로, 이를 고려하여 하나의 중합효소연쇄반응법 반응물에 함께 사용하는 형광 표지 인자는 별개로 검출가능한지 여부를 판단하여 선택 사용하여야 하며, 서로 다른 색상을 사용할 수 있다. 상기 형광 표지 인자에 대한 구체적인 사항 및 선택은 본원 발명에 속하는 기술분야의 당업자들에게 자명한 것이다.The anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 9 and the Mycobacterium tuberculosis IS6110 gene detection probe of SEQ ID NO: 3 may be labeled with different detectable means. The detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 포함한다. 상기 결핵균의 IS6110 유전자의 프라이머 세트는 검출 대상이 되는 여러 종의 결핵균(Mycobacterium tuberculosis complex, MTC)을 모두 검출할 수 있도록 설계되었다.The Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 1 and a reverse primer of nucleotide sequence 2. The primer set of the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes ( MTCs ).
본 발명의 일실시예에 따르면, 상기 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브와 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to one embodiment of the invention, the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3 and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 9 may be a Taqman probe.
상기 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브는 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3 is specific for the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2 Probe.
일실시예에 따르면, 상기 염기서열 4의 정방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 정방향 프라이머이다. 상기 염기서열 4(5′-ggyrayctgccctgcac-3′)의 경우, 5′-ggtaatctgccctgcac-3′ (염기서열 12), 5′-ggtaacctgccctgcac-3′ (염기서열 13), 5′-ggcaatctgccctgcac-3′ (염기서열 14) 및 5′-ggcaacctgccctgcac-3′ (염기서열 15), 5′-ggtgatctgccctgcac-3′ (염기서열 16), 5′-ggtgacctgccctgcac-3′ (염기서열 17), 5′-ggcgatctgccctgcac-3′ (염기서열 18) 및 5′-ggcgacctgccctgcac-3′ (염기서열 19)를 포함하는 프라이머 세트일 수 있다.예를 들어, 5′-ggtaatctgccctgcac-3′, 5′-ggtaacctgccctgcac-3′, 5′-ggcaatctgccctgcac-3′ 및 5′-ggcaacctgccctgcac-3′, 5′-ggtgatctgccctgcac-3′, 5′-ggtgacctgccctgcac-3′, 5′-ggcgatctgccctgcac-3′ 및 5′-ggcgacctgccctgcac-3′를 약 1:1:1:1:1:1:1:1의 비율로 포함되는 프라이머 세트일 수 있다.According to one embodiment, the forward primer of SEQ ID NO: 4 is 16S rRNA gene specific forward primer of non-acidic tuberculosis bacteria. In case of the nucleotide sequence 4 (5′-ggyrayctgccctgcac-3 ′), 5′-ggtaatctgccctgcac-3 ′ (base sequence 12), 5′-ggtaacctgccctgcac-3 ′ (base sequence 13), 5′-ggcaatctgccctgcac-3 ′ ( SEQ ID NO: 14) and 5′-ggcaacctgccctgcac-3 ′ (SEQ ID NO: 15), 5′-ggtgatctgccctgcac-3 ′ (SEQ ID NO: 16), 5′-ggtgacctgccctgcac-3 ′ (SEQ ID NO: 17), 5′-ggcgatctgccctgcac-3 Primer set comprising ′ (base 18) and 5′-ggcgacctgccctgcac-3 ′ (base 19). For example, 5′-ggtaatctgccctgcac-3 ′, 5′-ggtaacctgccctgcac-3 ′, 5 ′. -ggcaatctgccctgcac-3 ′ and 5′-ggcaacctgccctgcac-3 ′, 5′-ggtgatctgccctgcac-3 ′, 5′-ggtgacctgccctgcac-3 ′, 5′-ggcgatctgccctgcac-3 ′ and 5′-ggcgacctgccctgcac-3 ′ The primer set may be included in a ratio of 1: 1: 1: 1: 1: 1: 1.
상기 염기서열 5의 프라이머(NTM-1), 염기서열 6의 프라이머(NTM-1) 및 염기서열 7의 프라이머(NTM-1)는 16S rRNA 유전자 특이적 역방향 프라이머이다. 염기서열 8의 프라이머(NTM-2)는 항산성비결핵균의 16S rRNA 유전자 특이적 역방향 프라이머이다. 상기 항산성비결핵균의 16S rRNA 유전자의 역방향 프라이머는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다. 상기 염기서열 8(5′-catcccacaccgctaccw-3′)의 경우, 5′-catcccacaccgctacct-3′ (염기서열 10)과 5′-catcccacaccgctacca-3′ (염기서열 11)을 포함하는 프라이머 세트를 의미한다. 예를 들어, 염기서열 8의 프라이머는 5′-catcccacaccgctacct-3′와 5′-catcccacaccgctacca-3′를 약 1:1로 포함하는 프라이머 세트일 수 있다.The primer of SEQ ID NO: 5 (NTM-1), primer of SEQ ID NO: 6 (NTM-1) and primer of SEQ ID NO: 7 (NTM-1) are 16S rRNA gene specific reverse primers. A primer of SEQ ID NO: 8 (NTM-2) is a 16S rRNA gene specific reverse primer of non-acidic Mycobacterium tuberculosis. The reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected. In the case of the nucleotide sequence 8 (5′-catcccacaccgctaccw-3 ′), it means a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11). For example, the primer of SEQ ID NO: 8 may be a primer set including about 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ at about 1: 1.
본 발명의 일 실시예에 따르면, 상기 결핵균 IS6110 유전자 탐지용 프로브 및 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 표지되고, 상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다. 일 예로서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 VIC, 3′ 말단이 MGBNFQ으로 표지되고, 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, 3′ 말단이 MGBNFQ이 표시될 수 있다.According to an embodiment of the present invention, the 5 ′ end of the tuberculosis bacteria IS6110 gene detection probe and the anti-acidic tuberculosis 16S rRNA gene detection probe is FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS Labeled with one fluorescent labeling factor selected from the group consisting of RED, RED670 and NED, and with one fluorescent inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ at the 3 ′ end. The label is characterized in that, the 5 'end of the probe for detecting the IS6110 gene of the Mycobacterium tuberculosis and 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors. As an example, the 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe is labeled with VIC, and the 3' end is labeled with MGBNFQ, and the 5 'end of the FDR 16S rRNA gene detection probe is labeled with FAM, and the 3' end is labeled with MGBNFQ. Can be.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 5의 역방향 프라이머와 염기서열 8의 역방향 프라이머는 1:1일 수 있으며, 염기서열 8의 역방향 프라이머는 염기서열 10의 역방향 프라이머 및 염기서열 11의 역방향 프라이머를 1:1로 포함할 수 있다. 이에 따라, 염기서열 5의 프라이머, 5′-catcccacaccgctacct-3′ 및 5′-catcccacaccgctacca-3′은 2:1:1의 비를 가질 수 있다.According to one embodiment, in the Mycobacterium tuberculosis bacillus and anti-acidic tuberculosis detection kit, the reverse primer of the nucleotide sequence 5 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1. Accordingly, the primers 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ of base sequence 5 may have a ratio of 2: 1: 1.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 6의 역방향 프라이머와 염기서열 8의 역방향 프라이머는 1:1일 수 있으며, 염기서열 8의 역방향 프라이머는 염기서열 10의 역방향 프라이머 및 염기서열 11의 역방향 프라이머를 1:1로 포함할 수 있다.According to one embodiment, in the Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, the reverse primer of the base sequence 6 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 7의 역방향 프라이머와 염기서열 8의 역방향 프라이머는 1:1일 수 있으며, 염기서열 8의 역방향 프라이머는 염기서열 10의 역방향 프라이머 및 염기서열 11의 역방향 프라이머를 1:1로 포함할 수 있다.According to one embodiment, in the Mycobacterium tuberculosis bacillus and the anti-acidic non-tuberculosis bacterium detection kit, the reverse primer of the nucleotide sequence 7 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1.
상기 또 다른 목적을 달성하기 위하여, 본 발명은 검체시료로부터 DNA를 분리하는 단계; 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 4의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머(NTM-1) 및 염기서열 8의 역방향 프라이머(NTM-2)를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the above another object, the present invention comprises the steps of: separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; One or more reverse primers (NTM-1) selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7, and a reverse primer of nucleotide sequence 8 (NTM- A primer set specific for the 16S rRNA gene of anti-acidic Mycobacterium tuberculosis comprising 2); And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-tuberculosis bacterium 16S rRNA gene of SEQ ID NO: 9; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
상기 목적을 달성하기 위하여, 염기서열 22의 정방향 프라이머; 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머; 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 제공한다.In order to achieve the above object, a forward primer of SEQ ID NO: 22; One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And it provides a primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
상기 염기서열 22의 정방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 정방향 프라이머이다.The forward primer of SEQ ID NO: 22 is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis.
상기 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머는 16S rRNA 유전자 특이적 역방향 프라이머(NTM-1)이다. 염기서열 8의 프라이머(NTM-2)는 항산성비결핵균의 16S rRNA 유전자 특이적 역방향 프라이머이다. 상기 항산성비결핵균의 16S rRNA 유전자의 역방향 프라이머는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다. 상기 염기서열 8(5′-catcccacaccgctaccw-3′)의 경우, 5′-catcccacaccgctacct-3′ (염기서열 10)과 5′-catcccacaccgctacca-3′ (염기서열 11)을 포함하는 프라이머 세트로서, 염기서열 10과 염기서열 11을 약 1:1로 포함하는 프라이머 세트일 수 있다.The primer of SEQ ID NO: 5, primer of SEQ ID NO: 6, and primer of SEQ ID NO: 7 are 16S rRNA gene specific reverse primer (NTM-1). A primer of SEQ ID NO: 8 (NTM-2) is a 16S rRNA gene specific reverse primer of non-acidic Mycobacterium tuberculosis. The reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected. In the case of the nucleotide sequence 8 (5′-catcccacaccgctaccw-3 ′), a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11) It may be a primer set including about 10 and the base sequence 11 in about 1: 1.
본 발명의 목적을 달성하기 위하여, 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다.In order to achieve the object of the present invention, it provides a probe for detecting the acidic non-tuberculosis 16S rRNA gene of SEQ ID NO: 23.
본 발명의 일실시예에 따르면 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지될 수 있다.According to an embodiment of the present invention, the 5 ′ end of the probe is one fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a factor, and the 3 'end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3.
상기 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 23 is one or more reverse primers selected from the group consisting of a forward primer of SEQ ID NO: 22, a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7 And it is a probe specific to the reaction product of the polymerase chain reaction method using a primer specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of SEQ ID NO: 8.
상기 다른 목적을 달성하기 위하여, 본 발명은 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.In order to achieve the above another object, the present invention provides a primer set specific to the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And it provides a tuberculosis bacteria and anti-acidic tuberculosis bacteria detection kit comprising a probe for detecting the non-acidic tuberculosis 16S rRNA gene of SEQ ID NO: 23.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
상기 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다. 상기 검출가능한 수단은 프로브에 연결, 결합, 또는 부착시켜 통상적인 방식으로 밀도, 농도, 양 등을 확인할 수 있는 화합물, 생체 분자 또는 생체 분자 모방체 등을 의미한다. 그 예로, 통상적으로 사용되는 형광 표지인자, 발광물질, 생발광물질, 동위원소 등이 있으나, 이로 한정되는 것은 아니다. 형광 표지 인자는 종류에 따라 여기 및 방사 파장이 다르면 사용방법 또한 상이하므로, 이를 고려하여 하나의 중합효소연쇄반응법 반응물에 함께 사용하는 형광 표지 인자는 별개로 검출가능한지 여부를 판단하여 선택 사용하여야 하며, 서로 다른 색상을 사용할 수 있다. 상기 형광 표지 인자에 대한 구체적인 사항 및 선택은 본원 발명에 속하는 기술분야의 당업자들에게 자명한 것이다.The anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 23 and the Mycobacterium tuberculosis IS6110 gene detection probe of SEQ ID NO: 21 may be labeled with different detectable means. The detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 포함한다. 상기 결핵균의 IS6110 유전자의 프라이머는 검출 대상이 되는 여러 종의 결핵균(MTC)을 모두 검출할 수 있도록 설계되었다.The Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20. The primer of the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all kinds of Mycobacterium tuberculosis (MTC).
일 실시예에 따르면, 상기 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브와 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to one embodiment, the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe of SEQ ID NO: 23 may be a Taqman probe.
상기 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브는 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20 Probe.
상기 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머는 16S rRNA 유전자 특이적 역방향 프라이머(NTM-1)이다. 염기서열 8의 프라이머(NTM-2)는 항산성비결핵균의 16S rRNA 유전자 특이적 역방향 프라이머이다. 상기 항산성비결핵균의 16S rRNA 유전자의 역방향 프라이머는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다. 상기 염기서열 8(5′-catcccacaccgctaccw-3′)의 경우, 5′-catcccacaccgctacct-3′ (염기서열 10)과 5′-catcccacaccgctacca-3′ (염기서열 11)을 포함하는 프라이머 세트를 의미한다. 예를 들어, 염기서열 8의 프라이머는 5′-catcccacaccgctacct-3′와 5′-catcccacaccgctacca-3′를 약 1:1로 포함하는 프라이머 세트일 수 있다.The primer of SEQ ID NO: 5, primer of SEQ ID NO: 6, and primer of SEQ ID NO: 7 are 16S rRNA gene specific reverse primer (NTM-1). A primer of SEQ ID NO: 8 (NTM-2) is a 16S rRNA gene specific reverse primer of non-acidic Mycobacterium tuberculosis. The reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected. In the case of the nucleotide sequence 8 (5′-catcccacaccgctaccw-3 ′), it means a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11). For example, the primer of SEQ ID NO: 8 may be a primer set including about 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ at about 1: 1.
본 발명의 일실시예에 따르면, 상기 결핵균 IS6110 유전자 탐지용 프로브 및 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 표지되고, 상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다. 일 예로서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 HEX, 3′ 말단이 BHQ-1으로 표지되고, 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, 3′ 말단이 BHQ-1이 표시될 수 있다.According to one embodiment of the invention, the 5 'end of the tuberculosis bacteria IS6110 gene detection probe and the anti-acidic tuberculosis bacterium 16S rRNA gene detection probe is FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS Labeled with one fluorescent labeling agent selected from the group consisting of RED, RED670, and NED, and labeled with one fluorescent inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3 at the 3 ′ end. The 5 'end of the probe for detecting the IS6110 gene of the Mycobacterium tuberculosis bacterium and the 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors. As an example, the 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe is labeled with HEX and the 3' end is BHQ-1, and the 5 'end of the FSA 16S rRNA gene detection probe is FAM and the 3' end is BHQ. -1 may be displayed.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 5의 역방향 프라이머와 염기서열 8의 역방향 프라이머는 1:1일 수 있으며, 염기서열 8의 역방향 프라이머는 염기서열 10의 역방향 프라이머 및 염기서열 11의 역방향 프라이머를 1:1로 포함할 수 있다. 이에 따라, 염기서열 5의 프라이머, 5′-catcccacaccgctacct-3′ 및 5′-catcccacaccgctacca-3′은 2:1:1의 비를 가질 수 있다.According to one embodiment, in the Mycobacterium tuberculosis bacillus and anti-acidic tuberculosis detection kit, the reverse primer of the nucleotide sequence 5 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1. Accordingly, the primers 5′-catcccacaccgctacct-3 ′ and 5′-catcccacaccgctacca-3 ′ of base sequence 5 may have a ratio of 2: 1: 1.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 6의 역방향 프라이머와 염기서열 8의 역방향 프라이머는 1:1일 수 있으며, 염기서열 8의 역방향 프라이머는 염기서열 10의 역방향 프라이머 및 염기서열 11의 역방향 프라이머를 1:1로 포함할 수 있다.According to one embodiment, in the Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, the reverse primer of the base sequence 6 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 7의 역방향 프라이머와 염기서열 8의 역방향 프라이머는 1:1일 수 있으며, 염기서열 8의 역방향 프라이머는 염기서열 10의 역방향 프라이머 및 염기서열 11의 역방향 프라이머를 1:1로 포함할 수 있다.According to one embodiment, in the Mycobacterium tuberculosis bacillus and the anti-acidic non-tuberculosis bacterium detection kit, the reverse primer of the nucleotide sequence 7 and the reverse primer of the nucleotide sequence 8 may be 1: 1, the reverse primer of the nucleotide sequence 8 is the reverse primer of the nucleotide sequence 10 and Reverse primer of SEQ ID NO: 11 may be included 1: 1.
상기 또 다른 목적을 달성하기 위하여, 검체시료로부터 DNA를 분리하는 단계; 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the another object, the step of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 23; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
상기 목적을 달성하기 위하여, 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다.In order to achieve the above object, the present invention provides a probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26.
본 발명의 일실시예에 따르면, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지될 수 있다.According to one embodiment of the invention, the 5 'terminal of the probe is one of the fluorescence selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED It is labeled with a labeling factor, and the 3 'end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
상기 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머 세트를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머를 이용한 중합효소연쇄반응법의 반응산물 중 결핵균의 반응산물에 특이적인 프로브이다.The probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26 is a polymerase chain using a common primer for amplifying 16S rRNA genes of Mycobacterium tuberculosis and anti-acidic tuberculosis comprising a forward primer set of SEQ ID NO: 24 and a reverse primer set of 25 It is a probe specific for the reaction product of Mycobacterium tuberculosis among the reaction products of the reaction method.
상기 목적을 달성하기 위하여, 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다.In order to achieve the above object, it provides a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 28.
상기 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머 세트를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머를 이용한 중합효소연쇄반응법의 반응산물 중 항산성비결핵균의 반응산물에 특이적인 프로브이다.The probe for detecting an acidic non-tuberculosis 16S rRNA gene comprising the probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 28 is a 16S rRNA of Mycobacterium tuberculosis and antiacidic Mycobacterium tuberculosis comprising a forward primer set of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25 It is a probe specific to the reaction product of non-acidic tuberculosis bacteria among the reaction products of the polymerase chain reaction method using a common primer for amplifying a gene.
본 발명의 일실시예에 따르면, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지될 수 있다.According to one embodiment of the invention, the 5 'terminal of the probe is one of the fluorescence selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED It is labeled with a labeling factor, and the 3 'end may be labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
상기 다른 목적을 달성하기 위하여, 본 발명은 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트; 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브; 및 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.In order to achieve the above another object, the present invention provides a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and anti-acidic tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic tuberculosis 16S rRNA gene comprising a probe of the base sequence 27 and the probe of the base sequence 28.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
상기 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다. 상기 검출가능한 수단은 프로브에 연결, 결합, 또는 부착시켜 통상적인 방식으로 밀도, 농도, 양 등을 확인할 수 있는 화합물, 생체 분자 또는 생체 분자 모방체 등을 의미한다. 그 예로, 통상적으로 사용되는 형광 표지인자, 발광물질, 생발광물질, 동위원소 등이 있으나, 이로 한정되는 것은 아니다. 형광 표지 인자는 종류에 따라 여기 및 방사 파장이 다르면 사용방법 또한 상이하므로, 이를 고려하여 하나의 중합효소연쇄반응법 반응물에 함께 사용하는 형광 표지 인자는 별개로 검출가능한지 여부를 판단하여 선택 사용하여야 하며, 서로 다른 색상을 사용할 수 있다. 상기 형광 표지 인자에 대한 구체적인 사항 및 선택은 본원 발명에 속하는 기술분야의 당업자들에게 자명한 것이다.Probe for detection of Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26 and probe for detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising the probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 28 may be labeled with different detectable means. The detectable means means compounds, biomolecules or biomolecule mimetics, etc., which can be linked, bound, or attached to a probe to determine the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머는 마이코박테리아의 16S rRNA 유전자 부위를 증폭할 수 있는 프라이머로서, 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트이다.The forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 25 are primers capable of amplifying the 16S rRNA gene region of mycobacteria, and are a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis.
일 실시예에 따르면, 염기서열 24의 정방향 프라이머는 5′-ggataagcctgggaaactgg-3′ (염기서열 29)과 5′-ggataagcttgggaaactgg-3′ (염기서열 30)이 약 1:1의 비율로 포함되는 프라이머 세트일 수 있다. 또한, 염기서열 25의 역방향 프라이머는 5′-accccaccaacaagctgata-3′ (염기서열 31)과 5′-accccaccaactagctgata-3′ (염기서열 32)이 약 1:1의 비율로 포함되는 프라이머 세트일 수 있다.According to an embodiment, the forward primer of SEQ ID NO: 24 has a primer set including 5′-ggataagcctgggaaactgg-3 ′ (base sequence 29) and 5′-ggataagcttgggaaactgg-3 ′ (base sequence 30) in a ratio of about 1: 1 Can be. In addition, the reverse primer of SEQ ID NO: 25 may be a primer set containing 5′-accccaccaacaagctgata-3 ′ (base sequence 31) and 5′-accccaccaactagctgata-3 ′ (base sequence 32) in a ratio of about 1: 1.
일 실시예에 따르면, 상기 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to one embodiment, the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26, the probe of SEQ ID NO: 27, and the probe of SEQ ID NO: 28 may be a Taqman probe.
상기 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 결핵균(MTC)과 항산성비결핵균(NTM)을 특이적으로 구별할 수 있는 16S rRNA 염기부위을 타겟이 되도록 설계되었다.The non-acidic non-tuberculosis bacterium 16S rRNA gene detection probe comprising a probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO. It was designed to target specifically distinguishable 16S rRNA bases.
일 실시예에 따르면, NTM-2 프로브인 염기서열 28의 프로브는 5′-FAM-tggaaagcgtttggtagc-MGB-3′(염기서열 33)과 5′-FAM-tggaaagtgtttggtagc-MGB-3′ (염기서열 34)이 약 1:1 포함할 수 있다.According to one embodiment, the NTM-2 probe, the probe of SEQ ID NO: 28 is 5′-FAM-tggaaagcgtttggtagc-MGB-3 ′ (base sequence 33) and 5′-FAM-tggaaagtgtttggtagc-MGB-3 ′ (SEQ ID NO: 34) This may include about 1: 1.
본 발명의 일실시예에 따르면, 상기 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 표지되고, 상기 염기서열 26의 결핵균의 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다. 일 예로서, 상기 결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 VIC, 3′ 말단이 MGBNFQ으로 표지되고, 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, 3′ 말단이 MGBNFQ이 표시될 수 있다.According to an embodiment of the present invention, the 5 ′ terminal of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising the probe for detecting the Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26, the probe of SEQ ID NO: 27, and the probe of SEQ ID NO: 28 This is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED, and the 3 'end is 6-TAMRA, BHQ Labeled with one fluorescence inhibitor selected from the group consisting of -1,2,3 and MGBNFQ, and a probe for detecting 16S rRNA gene of Mycobacterium tuberculosis bacterium of SEQ ID NO: 26 and a probe and base of SEQ ID NO: 27; The 5 'end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising the probe of SEQ ID NO: 28 and the 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors. . As an example, the 5 'end of the Mycobacterium tuberculosis 16S rRNA gene detection probe is labeled with VIC, and the 3' end is labeled with MGBNFQ, and the 5 'end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe is labeled with FAM and the 3' end is MGBNFQ. Can be displayed.
상기 또 다른 목적을 달성하기 위하여, 검체시료로부터 DNA를 분리하는 단계; 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트; 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브; 및 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the another object, the step of separating the DNA from the sample; A common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
상기 목적을 달성하기 위하여, 본 발명은 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 제공한다.In order to achieve the above object, the present invention provides a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of nucleotide sequence 37, a probe of nucleotide sequence 38 and a probe of nucleotide sequence 39.
본 발명의 일실시예에 따르면, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ(molecular grove binding non-fluorescence quencher)로 이루어진 군으로부터 선택된 1종의 형광 억제 물질(Quencher)로 표지될 수 있다.According to one embodiment of the invention, the 5 'terminal of the probe is one of the fluorescence selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED Labeled with a marker factor, the 3 ′ end may be labeled with one fluorescence inhibitor (Quencher) selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ (molecular grove binding non-fluorescence quencher) have.
염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.Probe for detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a sequence of SEQ ID NO: 39 is a term comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36 It is a probe specific to the reaction product of the polymerase chain reaction method using a primer set specific for 16S rRNA gene of acidic mycobacterium tuberculosis.
상기 다른 목적을 달성하기 위하여, 본 발명은 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출키트를 제공한다.In order to achieve the above another object, the present invention provides a primer set specific to the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And it provides a tuberculosis bacteria and anti-acidic tuberculosis bacteria detection kit comprising a probe for detecting the non-acidic tuberculosis 16S rRNA gene selected from the group consisting of a probe of the nucleotide sequence 37, the probe of the nucleotide sequence 38 and the nucleotide sequence 39.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
염기서열 37의 프로브 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브와 상기 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다. 상기 검출 가능한 수단은 프로브에 연결, 결합, 또는 부착시켜 통상적인 방식으로 밀도, 농도, 양 등을 확인할 수 있는 화합물, 생체 분자 또는 생체 분자 모방체 등을 의미한다. 그 예로, 통상적으로 사용되는 형광 표지인자, 발광물질, 생발광물질, 동위원소 등이 있으나, 이로 한정되는 것은 아니다. 형광 표지 인자는 종류에 따라 여기 및 방사 파장이 다르면 사용방법 또한 상이하므로, 이를 고려하여 하나의 중합효소연쇄반응법 반응물에 함께 사용하는 형광 표지 인자는 별개로 검출가능한지 여부를 판단하여 선택 사용하여야 하며, 서로 다른 색상을 사용할 수 있다. 상기 형광 표지 인자에 대한 구체적인 사항 및 선택은 본원 발명에 속하는 기술분야의 당업자들에게 자명한 것이다.Probe of SEQ ID NO: 37 The probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of the probe of SEQ ID NO: 38 and the probe of SEQ ID NO: 39 and the probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 are different detectable means. Can be labeled. The detectable means means a compound, a biomolecule, or a biomolecule mimetic that can be linked, coupled, or attached to a probe to confirm the density, concentration, amount, etc. in a conventional manner. Examples thereof include fluorescent labeling factors, luminescent materials, bioluminescent materials, and isotopes that are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type of fluorescent labeling factor, the method of use is also different. Therefore, the fluorescent labeling factors used together in one polymerase chain reaction product should be selected and used to determine whether they are detectable separately. , Different colors are available. Specific details and selections for the fluorescent labeling factors will be apparent to those skilled in the art to which the present invention pertains.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 포함한다. 상기 결핵균의 IS6110 유전자의 프라이머 세트는 검출 대상이 되는 여러 종의 결핵균(Mycobacterium tuberculosis complex, MTC)을 모두 검출할 수 있도록 설계되었다.The Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20. The primer set of the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes ( MTCs ).
본 발명의 일실시예에 따르면, 상기 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브와 상기 염기서열 37의 프로브 염기서열, 38의 프로브 및 염기서열 39의 프로브로 이루어진 군에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to an embodiment of the present invention, the anti-acidic Mycobacterium tuberculosis 16S rRNA selected from the group consisting of the probe for detecting the Mycobacterium tuberculosis IS6110 gene of the nucleotide sequence 21 and the probe nucleotide sequence of the nucleotide sequence 37, the 38 probe, and the probe of the nucleotide sequence 39 The gene detection probe may be a Taqman probe.
상기 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브는 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20 Probe.
상기 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.Probe for detection of non-acidic mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of nucleotide sequence 37, a probe of nucleotide sequence 38 and a nucleotide sequence 39 comprises a forward primer of nucleotide sequence 35 and a reverse primer of nucleotide sequence 36 A probe specific for the reaction product of the polymerase chain reaction method using a primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacteria.
염기서열 35의 정방향 프라이머는 항산성비결핵균 16S rRNA 유전자 특이적 정방향 프라이머이다. 상기 항산성비결핵균의 16S rRNA 유전자의 정방향 프라이머는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다. 상기 염기서열 35의 경우, 5′-catgtcttgtgggggaaagctt-3′ (염기서열 40), 5′-catgttttgtgggggaaagctt-3′ (염기서열 41), 5′-catgtcttctgggggaaagctt-3′ (염기서열 42), 5′-catgtcttgtggtggaaagctt-3′ (염기서열 43), 5′-catgtcttgtggggcaaagctt-3′ (염기서열 44), 5′-catgttttctgggggaaagctt-3′ (염기서열 45), 5′-catgtcttctggtggaaagctt-3′ (염기서열 46), 5′-catgtcttgtggtgcaaagctt-3′ (염기서열 47), 5′-catgttttgtggggcaaagctt-3′ (염기서열 48), 5′-catgtcttctggggcaaagctt-3′ (염기서열 49), 5′-catgttttgtggtggaaagctt-3′ (염기서열 50), 5′-catgttttctggtggaaagctt-3′ (염기서열 51), 5′-catgttttctggggcaaagctt-3′ (염기서열 52), 5′-catgttttgtggtgcaaagctt-3′ (염기서열 53), 5′-catgtcttctggtgcaaagctt-3′ (염기서열 54) 및 5′-catgttttctggtgcaaagctt-3′ (염기서열 55)를 포함하는 프라이머 세트를 의미한다. 예를 들어, 염기서열 35의 프라이머는 염기서열 40 내지 55의 프라이머를 각각 실질적으로 동량 포함하는 프라이머 세트일 수 있다.The forward primer of SEQ ID NO: 35 is an anti-acidic Mycobacterium tuberculosis 16S rRNA gene specific forward primer. The forward primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected. For the base sequence 35, 5′-catgtcttgtgggggaaagctt-3 ′ (base sequence 40), 5′-catgttttgtgggggaaagctt-3 ′ (base sequence 41), 5′-catgtcttctgggggaaagctt-3 ′ (base sequence 42), 5′-catgtcttgtggtggaaagctt -3 '(base 43), 5'-catgtcttgtggggcaaagctt-3' (base 44), 5'-catgttttctgggggaaagctt-3 '(base 45), 5'-catgtcttctggtggaaagctt-3' (base 46), 5 ' -catgtcttgtggtgcaaagctt-3 ′ (base sequence 47), 5′-catgttttgtggggcaaagctt-3 ′ (base sequence 48), 5′-catgtcttctggggcaaagctt-3 ′ (base sequence 49), 5′-catgttttgtggtggaaagctt-3 ′ (base sequence 50), 5′-catgttttctggtggaaagctt-3 ′ (base sequence 51), 5′-catgttttctggggcaaagctt-3 ′ (base sequence 52), 5′-catgttttgtggtgcaaagctt-3 ′ (base sequence 53), 5′-catgtcttctggtgcaaagctt-3 ′ (base sequence 54) ) And 5'-catgttttctggtgcaaagctt-3 '(SEQ ID NO: 55). For example, the primer of SEQ ID NO: 35 may be a primer set that includes substantially the same amount of primers of SEQ ID NO: 40 to 55, respectively.
본 발명의 일실시예에 따르면, 상기 결핵균 IS6110 유전자 탐지용 프로브 및 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다. 일 예로서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 HEX, 3′ 말단이 BHQ-1으로 표지되고, 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, 3′ 말단이 BHQ-1이 표시될 수 있다.According to one embodiment of the invention, the 5 'end of the tuberculosis bacteria IS6110 gene detection probe and the anti-acidic tuberculosis bacterium 16S rRNA gene detection probe is FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS Labeled with one fluorescent labeling agent selected from the group consisting of RED, RED670, and NED, and the 3 ′ end is labeled with one fluorescent inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3 In addition, the 5 ′ end of the probe for detecting the IS6110 gene of the Mycobacterium tuberculosis and the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe may be labeled with different fluorescent labeling factors. As an example, the 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe is labeled with HEX and the 3' end is BHQ-1, and the 5 'end of the FSA 16S rRNA gene detection probe is FAM and the 3' end is BHQ. -1 may be displayed.
일 실시예에 따르면, 상기 결핵균과 항산성비결핵균 검출 키트에서 염기서열 38의 16S rRNA 유전자 탐지용 프로브는 FAM-cctgagagggtgaccgg-BHQ1 (염기서열 56) 및 FAM-cctgagagggtgtccgg-BHQ1 (염기서열 57)이 약 1:1로 포함할 수 있다.According to one embodiment, the 16S rRNA gene detection probe of SEQ ID NO: 38 in the Mycobacterium tuberculosis and anti-acidic Mycobacterium tuberculosis detection kit is FAM-cctgagagggtgaccgg-BHQ1 (SEQ ID NO: 56) and FAM-cctgagagggtgtccgg-BHQ1 (SEQ ID NO: 57) is about 1 Can be included as: 1.
상기 또 다른 목적을 달성하기 위하여, 검체시료로부터 DNA를 분리하는 단계; 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the another object, the step of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
상기 목적을 달성하기 위하여, 염기서열 61의 정방향 프라이머; 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 제공한다.In order to achieve the above object, a base primer of SEQ ID NO: 61; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer comprising a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 8.
상기 염기서열 61의 정방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 정방향 프라이머이다.The forward primer of SEQ ID NO: 61 is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis.
상기 염기서열 5의 프라이머(NTM-1) 및 염기서열 8의 프라이머(NTM-2)는 16S rRNA 유전자 특이적 역방향 프라이머이다. 상기 항산성비결핵균의 16S rRNA 유전자의 역방향 프라이머는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다. 상기 염기서열 8(5′-catcccacaccgctaccw-3′)의 경우, 5′-catcccacaccgctacct-3′ (염기서열 10)과 5′-catcccacaccgctacca-3′ (염기서열 11)을 포함하는 프라이머 세트로서, 염기서열 10과 염기서열 11을 약 1:1로 포함하는 프라이머 세트일 수 있다.The primer of SEQ ID NO: 5 (NTM-1) and the primer of SEQ ID NO: 8 (NTM-2) are 16S rRNA gene specific reverse primers. The reverse primers of the 16S rRNA gene of the anti-acidic tuberculosis bacterium were designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected. In the case of the nucleotide sequence 8 (5′-catcccacaccgctaccw-3 ′), a primer set including 5′-catcccacaccgctacct-3 ′ (base sequence 10) and 5′-catcccacaccgctacca-3 ′ (base sequence 11) It may be a primer set including about 10 and the base sequence 11 in about 1: 1.
상기 다른 목적을 달성하기 위하여, 본 발명은 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.In order to achieve the above another object, the present invention provides a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
본 발명의 일실시예에 따르면, 상기 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브 및 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다. According to an embodiment of the present invention, the anti-acidic Mycobacterium tuberculosis 16S rRNA selected from Mycobacterium tuberculosis IS6110 gene detection probe selected from the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 60 and the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63 Gene detection probes may be labeled by different detectable means from each other.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 포함한다. 상기 결핵균의 IS6110 유전자에 특이적인 프라이머 세트는 검출 대상이 되는 여러 종의 결핵균(Mycobacterium tuberculosis complex, MTC)을 모두 검출할 수 있도록 설계되었다.The Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59. The primer set specific for the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes (MTCs).
상기 염기서열 21의 프로브 또는 염기서열 60의 프로브는 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 60 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59 Probe.
본 발명의 일실시예에 따르면, 상기 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to an embodiment of the present invention, the probe for detecting Mycobacterium tuberculosis IS6110 gene selected from the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 60 may be a Taqman probe.
상기 염기서열 61의 정방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 정방향 프라이머이다. 상기 염기서열 5의 프라이머(NTM-1) 및 염기서열 8의 프라이머(NTM-2)는 항산성비결핵균의 16S rRNA 유전자 특이적 역방향 프라이머이다.The forward primer of SEQ ID NO: 61 is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis. The primer of SEQ ID NO: 5 (NTM-1) and the primer of SEQ ID NO: 8 (NTM-2) are 16S rRNA gene specific reverse primers of non-acidic Mycobacterium tuberculosis.
상기 염기서열 62의 프로브 또는 염기서열 63의 프로브는 염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63 is a primer specific for 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer comprising SEQ ID NO: 61, a primer of SEQ ID NO: 5, and a primer of SEQ ID NO: 8 A probe specific to the reaction product of polymerase chain reaction using a set.
본 발명의 일 실시예에 따르면, 상기 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to an embodiment of the present invention, the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 62 or the probe of SEQ ID NO: 63 may be a Taqman probe.
본 발명의 일 실시예에 따르면, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다.According to an embodiment of the present invention, the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Is labeled with one fluorescent labeling factor, and the 3 'end is labeled with one fluorescent inhibitor which is selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ. The 5 ′ end of the detection probe is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3 ′ The terminal is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ, and the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene are detected. The 5 ′ end of the probe for labeled with different fluorescent labeling factors Can.
상기 또 다른 목적을 달성하기 위하여, 검체시료로부터 DNA를 분리하는 단계; 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the another object, the step of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 62 or a probe of SEQ ID NO: 63; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
상기 목적을 달성하기 위하여, 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머; 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 제공한다.In order to achieve the above object, a forward primer comprising a primer of nucleotide sequence 65, a primer of nucleotide sequence 66 and a primer of nucleotide sequence 67; And it provides a primer set specific to 16S rRNA gene of non-acidic tuberculosis bacterium comprising a reverse primer of nucleotide sequence 36.
상기 염기서열 65의 프라이머(NTM-1), 염기서열 66의 프라이머(NTM-2) 및 염기서열 67의 프라이머(NTM-3)를 포함하는 정방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 정방향 프라이머이다. 상기 항산성비결핵균의 16S rRNA 유전자 정방향 프라이머는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다. 상기 염기서열 65(5′-tktggtggaaagcttttgc-3′)의 경우, 5′-tgtggtggaaagcttttgc-3′(염기서열 69)과 5′-tttggtggaaagcttttgc-3′(염기서열 70)을 포함하는 프라이머 세트로서, 염기서열 69와 염기서열 70을 약 1:1로 포함하는 프라이머 세트일 수 있다. 상기 염기서열 66(5′-ggtgwgtggtgcaaagctt-3′)의 경우, 5′-ggtgagtggtgcaaagctt-3′(염기서열 71)과 5′-ggtgtgtggtgcaaagctt-3′(염기서열 72)을 포함하는 프라이머 세트로서, 염기서열 71과 염기서열 72를 약 1:1로 포함하는 프라이머 세트일 수 있다.The forward primer comprising the primer of SEQ ID NO: 65 (NTM-1), the primer of SEQ ID NO: 66 (NTM-2) and the primer of SEQ ID NO: 67 (NTM-3) is a 16S rRNA gene specific forward primer of non-acidic Mycobacterium tuberculosis. to be. The 16S rRNA gene forward primer of the anti-acidic tuberculosis bacterium was designed to detect all of the various types of anti-acidic tuberculosis bacteria to be detected. In the case of the nucleotide sequence 65 (5′-tktggtggaaagcttttgc-3 ′), it is a primer set including 5′-tgtggtggaaagcttttgc-3 ′ (base sequence 69) and 5′-tttggtggaaagcttttgc-3 ′ (base sequence 70). It may be a primer set comprising about 1: 1 and 69 and nucleotide sequence 70. In the case of the nucleotide sequence 66 (5′-ggtgwgtggtgcaaagctt-3 ′), a primer set including 5′-ggtgagtggtgcaaagctt-3 ′ (base sequence 71) and 5′-ggtgtgtggtgcaaagctt-3 ′ (base sequence 72) It may be a primer set including 71 and SEQ ID NO: 72 in about 1: 1.
상기 다른 목적을 달성하기 위하여, 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.In order to achieve the above another object, a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the probe of SEQ ID NO: 39 or the base sequence 68.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
본 발명의 일실시예에 따르면, 상기 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브 및 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다.According to one embodiment of the present invention, the anti-acidic mycobacterium tuberculosis 16S rRNA selected from the Mycobacterium tuberculosis IS6110 gene detection probe selected from the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 64 and the probe of SEQ ID NO: 39 or the probe of SEQ ID NO: 68 Gene detection probes may be labeled by different detectable means from each other.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 포함한다. 상기 결핵균의 IS6110 유전자에 특이적인 프라이머 세트는 검출 대상이 되는 여러 종의 결핵균(Mycobacterium tuberculosis complex, MTC)을 모두 검출할 수 있도록 설계되었다.The Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20. The primer set specific for the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes (MTCs).
상기 염기서열 21의 프로브 또는 염기서열 64의 프로브는 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 64 is specific for the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20 Probe.
본 발명의 일실시예에 따르면, 상기 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to one embodiment of the present invention, the probe for detecting Mycobacterium tuberculosis IS6110 gene selected from the probe of SEQ ID NO: 21 or the probe of SEQ ID NO: 64 may be a Taqman probe.
상기 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 정방향 프라이머이다. 상기 염기서열 36의 역방향 프라이머는 항산성비결핵균의 16S rRNA 유전자 특이적 역방향 프라이머이다.The forward primer comprising the primer of SEQ ID NO: 65, the primer of SEQ ID NO: 66, and the primer of SEQ ID NO: 67 is a 16S rRNA gene specific forward primer of anti-acidic Mycobacterium tuberculosis. The reverse primer of SEQ ID NO: 36 is a 16S rRNA gene specific reverse primer of non-acidic mycobacterium tuberculosis.
상기 염기서열 39의 프로브 또는 염기서열 68의 프로브는 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe of SEQ ID NO: 39 or the probe of SEQ ID NO: 68 is a 16S antifungal tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; and a reverse primer of SEQ ID NO: 36 Probe specific to the reaction product of the polymerase chain reaction method using a primer set specific to the rRNA gene.
본 발명의 일 실시예에 따르면, 상기 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균의 16S rRNA 유전자 탐지용 프로브는 Taqman 프로브일 수 있다. 상기 염기서열 68의 프로브(5′-FAM-cctgagagggtgwccg-MGB-3′)의 경우, 5′-FAM-cctgagagggtgaccg-MGB-3′ (염기서열 73)과 5′-FAM-cctgagagggtgtccg-MGB-3′ (염기서열 74)을 포함하는 프라이머 세트로서, 염기서열 73 및 염기서열 74를 1:1로 포함하는 프로브 세트일 수 있다.According to one embodiment of the present invention, the probe for detecting 16S rRNA gene of non-acidic Mycobacterium tuberculosis selected from the probe of SEQ ID NO: 39 or the probe of SEQ ID NO: 68 may be a Taqman probe. In the case of the probe of base sequence 68 (5′-FAM-cctgagagggtgwccg-MGB-3 ′), 5′-FAM-cctgagagggtgaccg-MGB-3 ′ (base sequence 73) and 5′-FAM-cctgagagggtgtccg-MGB-3 ′ As a primer set including (SEQ ID NO: 74), it may be a probe set including the base sequence 73 and the base sequence 74 in a 1: 1.
본 발명의 일 실시예에 따르면, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다.According to an embodiment of the present invention, the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Is labeled with one fluorescent labeling factor, and the 3 'end is labeled with one fluorescent inhibitor which is selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ. The 5 ′ end of the detection probe is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3 ′ The terminal is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ, and the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene are detected. The 5 ′ end of the probe for labeled with different fluorescent labeling factors Can.
상기 또 다른 목적을 달성하기 위하여, 검체시료로부터 DNA를 분리하는 단계; 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the another object, the step of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 39 or a probe of SEQ ID NO: 68; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
상기 다른 목적을 달성하기 위하여, 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트를 제공한다.In order to achieve the above another object, a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of nucleotide sequence 58 and a reverse primer of nucleotide sequence 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And it provides a tuberculosis bacteria and anti-acidic tuberculosis detection kit comprising a probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76.
상기 검출 키트는 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약을 포함할 수 있다. 상기 DNA를 실시간 중합효소연쇄반응으로 증폭하기 위한 시약으로는 DNA 중합효소, dNTPs, PCR 버퍼 등을 포함할 수 있다.The detection kit may include a reagent for amplifying the DNA by real time polymerase chain reaction. Reagents for amplifying the DNA by real-time polymerase chain reaction may include DNA polymerase, dNTPs, PCR buffer, and the like.
본 발명의 일실시예에 따르면, 상기 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브 및 염기서열 27의 프로브(NTM-1) 및 염기서열 76의 프로브(NTM-2)를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 서로 상이한 검출 가능한 수단으로 표지될 수 있다.According to one embodiment of the present invention, the anti-acidic Mycobacterium tuberculosis 16S rRNA comprising the probe for detecting the Mycobacterium tuberculosis IS6110 gene of base sequence 60 and the probe of base sequence 27 (NTM-1) and the probe of base sequence 76 (NTM-2) Gene detection probes may be labeled by different detectable means from each other.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 포함한다. 상기 결핵균의 IS6110 유전자에 특이적인 프라이머 세트는 검출 대상이 되는 여러 종의 결핵균(Mycobacterium tuberculosis complex, MTC)을 모두 검출할 수 있도록 설계되었다.The Mycobacterium tuberculosis bacterium and the anti-acidic Mycobacterium tuberculosis detection kit include a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59. The primer set specific for the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the Mycobacterium tuberculosis complexes (MTCs).
상기 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브는 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60 is specific to the reaction product of the polymerase chain reaction method using a primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59 Probe.
본 발명의 일실시예에 따르면, 상기 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브는 Taqman 프로브일 수 있다.According to an embodiment of the present invention, the probe for detecting the Mycobacterium tuberculosis IS6110 gene of nucleotide sequence 60 may be a Taqman probe.
상기 결핵균과 항산성비결핵균 검출 키트는 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트를 포함한다. 상기 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머는 마이코박테리아의 16S rRNA 유전자 부위를 증폭할 수 있는 프라이머로서, 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트이다.The Mycobacterium tuberculosis and anti-acidic Mycobacterium tuberculosis detection kit includes a primer set specific for the 16S rRNA gene of Mycobacteria including a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75. The forward primer of SEQ ID NO: 24 and the reverse primer of SEQ ID NO: 75 are primers capable of amplifying the 16S rRNA gene region of mycobacteria, and are a common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis.
상기 염기서열 24의 정방향 프라이머는 마이코박테리아의 16S rRNA 유전자 특이적 정방향 프라이머이다. 상기 염기서열 24(5′-ggataagcytgggaaactgg-3′)의 경우, 5′-ggataagcctgggaaactgg-3′ (염기서열 29)과 5′-ggataagcttgggaaactgg-3′ (염기서열 30)을 포함하는 프라이머 세트로서, 염기서열 29와 염기서열 30을 약 1:1로 포함하는 프라이머 세트일 수 있다.The forward primer of SEQ ID NO: 24 is a 16S rRNA gene specific forward primer of mycobacteria. In the case of the nucleotide sequence 24 (5′-ggataagcytgggaaactgg-3 ′), the primer set includes 5′-ggataagcctgggaaactgg-3 ′ (base sequence 29) and 5′-ggataagcttgggaaactgg-3 ′ (base sequence 30). It may be a primer set including 29 and nucleotide sequence 30 in about 1: 1.
상기 염기서열 36의 역방향 프라이머는 마이코박테리아의 16S rRNA 유전자 특이적 역방향 프라이머이다.The reverse primer of SEQ ID NO: 36 is a 16S rRNA gene specific reverse primer of mycobacteria.
상기 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트를 이용한 중합효소연쇄반응법의 반응산물 중에서 항산성비결핵균의 중합효소연쇄반응법의 반응산물에 특이적인 프로브이다.The probe for detecting an acidic non-TB tuberculosis 16S rRNA gene comprising the probe of SEQ ID NO: 27 and the probe of SEQ ID NO: 76 is specific for the 16S rRNA gene of Mycobacteria including a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75 Among the reaction products of the polymerase chain reaction method using a primer set, it is a probe specific to the reaction product of the polymerase chain reaction method of non-acidic tuberculosis bacteria.
상기 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브는 검출 대상이 되는 여러 종의 항산성비결핵균을 모두 검출할 수 있도록 설계되었다.The anti-acidic tuberculosis 16S rRNA gene detection probe including the probe of SEQ ID NO: 27 and the probe of base sequence 76 was designed to detect all kinds of anti-acidic tuberculosis bacteria to be detected.
본 발명의 일 실시예에 따르면, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지될 수 있다.According to an embodiment of the present invention, the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Is labeled with one fluorescent labeling factor, and the 3 'end is labeled with one fluorescent inhibitor which is selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ. The 5 ′ end of the detection probe is labeled with one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED, 3 ′ The terminal is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ, and the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe and the anti-acidic Mycobacterium tuberculosis 16S rRNA gene are detected. The 5 ′ end of the probe for labeled with different fluorescent labeling factors Can.
상기 또 다른 목적을 달성하기 위하여, 검체시료로부터 DNA를 분리하는 단계; 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및 상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법을 제공한다.In order to achieve the another object, the step of separating the DNA from the sample; A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76; And it provides a method for detecting Mycobacterium tuberculosis and non-acidic tuberculosis bacteria comprising the step of confirming the double real-time polymerase chain reaction results.
이하, 본 발명을 참고예 및 실시예에 의해 상세히 설명하지만, 하기 참고예 및 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by reference examples and examples, but the following reference examples and examples are merely illustrative of the present invention, and the content of the present invention is not limited thereto.
참고예: 결핵균과 항산성비결핵균의 특이적 염기서열 탐색Reference Example: Searching for Specific Sequences of Mycobacterium Tuberculosis and Antiacidic Mycobacterium Tuberculosis
1. 대상 및 유전자 부위1. Target and Gene Site
결핵균과 항산성비결핵균의 특이적 염기서열 탐색에는 M. abscessus (AJ419970.1, AJ416940.1, AJ536038), M. acapulcensis (AF480575.1), M. africanum (AF480605.1), M. agri (AJ429045.1), M. aichiense (X55598.1), M. alvei (NR_024859.1), M. asiaticum (X55604.1), M. aurum (FJ172298.1), M. austroafricanum (GU121552.1), M. avium (NR_025584.1, AJ536037.1, EF521892.1), M. bohemicum (NR_026054.1), M. botniense (NR_028878.1), M. bovis (GU142937.1), M. branderi (AF480574.1), M. brumae (NR_025233.1), M. celatum (L08169.1), M.chelonae (AM884324.1, AJ419969.1), M. chitae (NR_029220.1), M. chlorophenolicum (NR_026173.1), M. chubuense (X55596.1), M. confluentis (AJ634379.1), M. conspicuum (X029298.1), M. cookii (X53896.1), M. diernhoferi (AF480599.1), M. doricum (NR_025099.1), M. duvalii (NR_026073.1), M. engbaekii (AF480577.1), M. fallax (AF480600.1), M. farcinogenes (X55592.1), M. flavescens (AY734993.1), M. fortuitum (AY457066.1, AF480580.1, GU142933.1), M. gadium (NR_026087.1), M. gastri (GU142918.1), M. genavense (NR_029223.1), M. gilvum (AB491971.1), M. goodii (AY457079.1), M. gordonae (GU142923.1), M. haemophilum (V06638.1), M. hassiacum (NR_026011.1), M. heidelbergense (NR_025268.1), M. hiberniae (NR_026092.1), M. hodleri (NR_026286.1), M. immunogen (AJ011771.1), M. interjectum (X70961.1), M. intermedium (X67847.1), M.intracellulare (AY652958.1, AJ536036.1, X52927.1, M61684.1), M.kansasii (M29575.1, X15916.1), M. lentiflavum (AF480583.1), M. mageritense (AY457076.1), M. malmoense (GQ153278.1), M. marinum (AF456238.1, AY513243.1), M. microti (NR_025234.1), M. monacense (GU142931.1), M. moriokaense (AY859686.1), M. mucogenicum (AF480585.1), M. neoaurum (FJ172306.1), M.nonchromogenicum (DQ058406.1), M. obuense (X55597.1), M. paraffinicum (GQ153282.1), M. parafortuitum (NR_026285.1), M. peregrinum (AY457069.1), M. phlei (AF480603.1), M. porcinum (AY457077.1), M. poriferae (NR_025235.1), M. pulveris (NR_025528.1), M. rhodesiae (NR_025529.1), M. scrofulaceum (GQ153271.1), M. senuense (DQ536409.1), M. septicum (AY457070.1), M. shimoidei (X82459.1), M. simiae (GQ153280.1), M. smegmatis (NR_025311.1), M. sphagni (X55590.1), M. szulgai (X52926.1), M. terrae (NR_029168.1), M. thermoresistibile (GU142928.1), M. tilburgii (AJ580826.1), M. triplex (GQ153279.1), M. triviale (DQ058405.1), M. tuberculosis (GU142936.1, GU142935.1, AY53603.1, X55588.1, X52917.1), M. tusciae (NR_024903.1), M. ulcerans (Z13990.1), M. vaccae (X55601.1), M. wolinskyi (AY457083.1), M. xenopi (X52929.1)의 16S ribosomal RNA 유전자의 염기서열 자료를 분석에 이용하였다. 상기 마이코박테리아의 16S ribosomal RNA 유전자의 염기서열 자료는 NCBI(National center for Biotechnology Information)의 데이터베이스(Databases)에서 얻었다 M. abscessus (AJ419970.1, AJ416940.1, AJ536038), M. acapulcensis (AF480575.1), M. africanum (AF480605.1), M. agri (AJ429045) .1), M. aichiense (X55598.1), M. alvei (NR_024859.1), M. asiaticum (X55604.1), M. aurum (FJ172298.1), M. austroafricanum (GU121552.1), M avium (NR_025584.1, AJ536037.1, EF521892.1), M. bohemicum (NR_026054.1), M. botniense (NR_028878.1), M. bovis (GU142937.1), M. branderi (AF480574.1) ), M. brumae (NR_025233.1), M. celatum (L08169.1), M.chelonae (AM884324.1, AJ419969.1), M. chitae (NR_029220.1), M. chlorophenolicum (NR_026173.1) , M. chubuense (X55596.1), M. confluentis (AJ634379.1), M. conspicuum (X029298.1), M. cookii (X53896.1), M. diernhoferi (AF480599.1), M. doricum ( NR_025099.1), M. duvalii (NR_026073.1), M. engbaekii (AF480577.1), M. fallax (AF480600.1) , M. farcinogenes (X55592.1), M. flavescens (AY734993.1), M. fortuitum (AY457066.1, AF480580.1, GU142933.1), M. gadium (NR — 026087.1), M. gastri (GU142918.1), M. genavense (NR_029223.1), M. gilvum (AB491971.1), M. goodii (AY457079.1), M. gordonae (GU142923.1), M. haemophilum (V06638.1) ), M. hassiacum (NR_026011.1), M. heidelbergense (NR_025268.1), M. hiberniae (NR_026092.1), M. hodleri (NR_026286.1), M. immunogen (AJ011771.1), M. interjectum (X70961.1), M. intermedium (X67847.1), M. intracellulare (AY652958.1, AJ536036.1, X52927.1, M61684.1), M.kansasii (M29575.1, X15916.1), M lentiflavum (AF480583.1), M. mageritense (AY457076.1), M. malmoense (GQ153278.1), M. marinum (AF456238.1, AY513243.1), M. microti (NR_025234.1), M. monacense (GU142931.1), M. moriokaense (AY859686.1), M. mucogenicum (AF480585.1), M. neoaurum (FJ172306.1), M. nonchromogenicum (DQ058406.1), M. obuense (X55597.1 ), M. paraffinicum (GQ153282.1), M. parafortuitum (NR_026285.1), M. peregrinum (AY457069.1) , M. phlei (AF480603.1), M. porcinum (AY457077.1), M. poriferae (NR_025235.1) , M. pulveris (NR_025528.1), M. rhodesiae (NR_025529.1), M. scrofulaceum (G Q153271.1), M. senuense (DQ536409.1) , M. septicum (AY457070.1), M. shimoidei (X82459.1), M. simiae (GQ153280.1), M. smegmatis (NR_025311.1), M. sphagni (X55590.1), M. szulgai (X52926.1), M. terrae (NR_029168.1), M. thermoresistibile (GU142928.1), M. tilburgii (AJ580826.1), M. triplex (GQ153279 .1), M. triviale (DQ058405.1), M. tuberculosis (GU142936.1, GU142935.1, AY53603.1, X55588.1, X52917.1), M. tusciae (NR_024903.1), M. ulcerans The base sequence data of 16S ribosomal RNA genes of (Z13990.1), M. vaccae (X55601.1) , M. wolinskyi (AY457083.1) and M. xenopi (X52929.1) were used for the analysis. Sequence data of the 16S ribosomal RNA gene of the mycobacteria was obtained from databases of the National Center for Biotechnology Information (NCBI).
상기 마이코박테리아 균종의 16S rRNA 유전자의 염기서열 자료를 Sequencher 4.9로 분석하여 특이 염기서열 부위를 발견하였다. 즉, 결핵균 특이적 염기서열 및 결핵균에는 없고 항산성비결핵균의 고유염기서열을 발견하였다.The nucleotide sequence data of the 16S rRNA gene of the Mycobacteria strain was analyzed by Sequencher 4.9 to find a specific sequence region. In other words, the nucleotide sequence specific to Mycobacterium tuberculosis and the native nucleotide sequence of mycobacterium tuberculosis were not found.
실시예 1: 결핵균과 항산성비결핵균의 분리검출 방법 1Example 1 Method for Separation and Detection of Mycobacterium Tuberculosis and Anti-acidic Tuberculosis
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
결핵균(Mycobacterium tuberculosis complex, MTC: M. tuberculosis, M.bovis, M. africanum, M. microti)은 IS6110 유전자를 검출 대상으로 하고, 항산성비결핵균(NTM)은 16S rRNA 유전자를 검출대상으로 하여 Taqman 프로브와 프라이머를 사용하였다. 상기 검출대상 부위용 프라이머는 Primer3 프로그램을 이용하여 설계하였다.Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe with 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
(1) 결핵균(MTC)(1) Mycobacterium tuberculosis (MTC)
1) 대상 유전자: IS61101) Target gene: IS6110
2) 프라이머2) primer
a. 정방향 프라이머: 5′-cgaactcaaggagcacatca-3′(염기서열 1)a. Forward primer: 5'-cgaactcaaggagcacatca-3 '(SEQ ID NO: 1)
b. 역방향 프라이머: 5′-agtttggtcatcagccgttc-3′(염기서열 2)b. Reverse primer: 5′-agtttggtcatcagccgttc-3 ′ (SEQ ID NO: 2)
3) Taqman 프로브3) Taqman Probe
5′-VIC-agtgtggctaaccctgaa-MGB-3′(염기서열 3)5'-VIC-agtgtggctaaccctgaa-MGB-3 '(SEQ ID NO: 3)
4) PCR 산물의 크기: 135bp4) Size of PCR product: 135bp
(2) 항산성비결핵균(NTM)(2) anti-acidic tuberculosis bacteria (NTM)
1) 대상유전자: 16S rRNA1) Target Gene: 16S rRNA
2) 프라이머2) primer
a. 정방향 프라이머: 5′-ggyrayctgccctgcac-3′(염기서열 4)a. Forward primer: 5'-ggyrayctgccctgcac-3 '(SEQ ID NO: 4)
b. 역방향 프라이머b. Reverse primer
NTM-1: 5′-cccacaccgcaaaagctt-3′ (염기서열 5) 또는 5′-cccacaccgcaaaagct-3′ (염기서열 6) 또는 5′-tcccacaccgcaaaagct-3′ (염기서열 7)NTM-1: 5′-cccacaccgcaaaagctt-3 ′ (base 5) or 5′-cccacaccgcaaaagct-3 ′ (base 6) or 5′-tcccacaccgcaaaagct-3 ′ (base 7)
NTM-2: 5′-catcccacaccgctaccw-3′(염기서열 8)NTM-2: 5′-catcccacaccgctaccw-3 ′ (SEQ ID NO: 8)
3) Taqman 프로브3) Taqman Probe
5′-FAM-cggtattagacccagtttcc-MGB-3′(염기서열 9)5′-FAM-cggtattagacccagtttcc-MGB-3 ′ (SEQ ID NO: 9)
4) PCR 산물의 크기: 104bp4) Size of PCR product: 104bp
<실시예 1-1. 항산성비결핵균 검출용 역방향 프라이머 NTM-1으로 염기서열 5의 프라이머를 사용하는 이중 실시간 중합효소연쇄반응법><Example 1-1. Double real-time polymerase chain reaction using a primer of SEQ ID NO: 5 as a reverse primer NTM-1 for detecting acidic non-TB bacteria>
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 7주를 사용하였다. 사용된 ATCC 표준균주는 M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus (ATCC 19977), M. avium (ATCC 25291)이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated. The ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 7주를 사용하였다. 사용된 ATCC 표준균주는 M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus (ATCC 19977), M. avium (ATCC 25291)이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated. The ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC 표준균주는 액체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC standard strain was used by culturing in a liquid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)를 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 15초 간 변성, 약 66℃에서 약 15초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 4와 같다. 하기 primer-probes Mix에는 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있고 프루브는 4pmole/㎕이 있었다. 따라서 반응에 사용되는 MTC의 primer-probes mix 1.25㎕에는 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 프로브는 5pmole이 들어 있게 되었다. 총 25uL의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmoles/25㎕), 프로브는 0.2uM(5pmole/25㎕)가 사용되었다. NTM의 정방향 및 역방향 프라이머, 프로브의 농도 및 부피는 MTC와 동일하게 사용되었다. NTM-1 역방향 프라이머로는 염기서열 5의 프라이머가 NTM-2 역방향 프라이머로는 염기서열 8의 프라이머가 동량으로 사용하였다. NTM-2 역방향 프라이머는 5′-catcccacaccgctacct-3′ (염기서열 10)와 5′-catcccacaccgctacca-3′ (염기서열 11)이 동량으로 존재하도록 설계되었다. NTM 정방향 프라이머는 5′-ggtaatctgccctgcac-3′ (염기서열 12), 5′-ggtaacctgccctgcac-3′ (염기서열 13), 5′-ggcaatctgccctgcac-3′ (염기서열 14), 5′-ggcaacctgccctgcac-3′ (염기서열 15), 5′-ggtgatctgccctgcac-3′ (염기서열 16), 5′-ggtgacctgccctgcac-3′ (염기서열 17), 5′-ggcgatctgccctgcac-3′ (염기서열 18) 및 5′-ggcgacctgccctgcac-3′ (염기서열 19)을 포함하는 프라이머 세트로서, 염기서열 12, 염기서열 13, 염기서열 14, 염기서열 15, 염기서열 16, 염기서열 17, 염기서열 18 및 염기서열 19의 프라이머를 각각 약 1:1:1:1:1:1:1:1의 비율로 포함하도록 제작되었다.Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were carried out using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 66 ° C. for about 15 seconds. At this time, the composition of the reactants performing the double real-time polymerase chain reaction is shown in Table 4. In the following primer-probes Mix, the forward primer and the reverse primer contained the same amount (10 pmole / μl) and the probe was 4 pmole / μl. Therefore, 1.25 μl of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole. The total volume of 25uL of polymerase chain reaction was 25µL. The primer concentration was 0.5uM (12.5pmoles / 25µl) and the probe 0.2uM (5 pmole/25µl). The concentration and volume of forward and reverse primers, probes of NTM were used the same as MTC. As the NTM-1 reverse primer, the primer of nucleotide sequence 5 was used as the NTM-2 reverse primer, and the primer of the nucleotide sequence 8 was used in the same amount. NTM-2 reverse primers were designed such that 5′-catcccacaccgctacct-3 ′ (SEQ ID NO: 10) and 5′-catcccacaccgctacca-3 ′ (SEQ ID NO: 11) are present in equal amounts. NTM forward primers are 5′-ggtaatctgccctgcac-3 ′ (SEQ ID NO: 12), 5′-ggtaacctgccctgcac-3 ′ (SEQ ID NO: 13), 5′-ggcaatctgccctgcac-3 ′ (SEQ ID NO: 14), 5′-ggcaacctgccctgcac-3 ′ (Base 15), 5′-ggtgatctgccctgcac-3 ′ (base 16), 5′-ggtgacctgccctgcac-3 ′ (base 17), 5′-ggcgatctgccctgcac-3 ′ (base 18) and 5′-ggcgacctgccctgcac- A primer set including 3 ′ (base sequence 19), wherein the primers of nucleotide sequence 12, nucleotide sequence 13, nucleotide sequence 14, nucleotide sequence 15, nucleotide sequence 16, nucleotide sequence 17, nucleotide sequence 18, and nucleotide sequence 19 are approximately It is designed to contain 1: 1: 1: 1: 1: 1: 1: 1.
표 1
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
Table 1
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), VICTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . On the real-time monitor, it was designated to display on the green channel (510 ± 5nm) when the FAM TM was developed and on the yellow channel (555 ± 5nm) when the VIC TM was developed. Fluorescence was observed in the green channel and the yellow channel.
<실시예 1-2. 항산성비결핵균 검출용 역방향 프라이머 NTM-1으로 염기서열 6의 프라이머를 사용하는 이중 실시간 중합효소연쇄반응법><Example 1-2. Double real-time polymerase chain reaction using the primer of nucleotide sequence 6 as the reverse primer NTM-1 for the detection of non-acidic tuberculosis bacteria>
실시예 1-1과 NTM-1의 역방향 프라이머로 염기서열 6의 프라이머를 사용하는 것을 제외하고는 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Except for using the primer of SEQ ID NO: 6 as the reverse primer of Example 1-1 and NTM-1, the double real-time polymerase chain reaction was carried out in substantially the same manner.
<실시예 1-3. 항산성비결핵균 검출용 역방향 프라이머 NTM-1으로 염기서열 7의 프라이머를 사용하는 이중 실시간 중합효소연쇄반응법><Example 1-3. Double real-time polymerase chain reaction using primers of SEQ ID NO: 7 as reverse primer NTM-1
실시예 1-1과 NTM-1의 역방향 프라이머로 염기서열 7의 프라이머를 사용하는 것을 제외하고는 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Except for using the primer of SEQ ID NO: 7 as the reverse primer of Example 1-1 and NTM-1, the double real-time polymerase chain reaction was carried out in substantially the same manner.
2. 이중 실시간 중합효소연쇄반응법 수행 결과2. Result of double real time polymerase chain reaction
도 1 내지 도 6는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 1 및 도 2는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 3 및 도 4는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 5 및 도 6는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.1 to 6 show the results obtained in the double real time polymerase chain reaction of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 1 and 2 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in the green and yellow channels of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. 3 and 4 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM). 5 and 6 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
도 1 내지 도 6를 참조하면, 결핵균(MTC)은 노랑채널에서 IS6110 유전자, 항산성비결핵균(NTM)은 녹색채널에서 16S rRNA 유전자, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 노랑채널과 녹색채널에서 IS6110 유전자와 16S rRNA 유전자가 특이적으로 증폭되는 결과를 나타내어, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.1 to 6, Mycobacterium tuberculosis (MTC) is the IS6110 gene in the yellow channel, acidic non-tuberculosis (NTM) is the 16S rRNA gene in the green channel, Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is the yellow channel and The results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel. When using the primers and probes according to the present invention, the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
실시예 2. 결핵균과 항산성비결핵균의 분리검출 방법 2Example 2 Separation and Detection Method of Mycobacterium Tuberculosis and Antimicrobial Mycobacterium Tuberculosis 2
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
결핵균(Mycobacterium tuberculosis complex, MTC: M. tuberculosis, M. bovis, M. africanum, M. microti)은 IS6110 유전자를 검출 대상으로 하고, 항산성비결핵균(NTM)은 16S rRNA 유전자를 검출대상으로 하여 Taqman 프로브와 프라이머를 사용하였다. 상기 검출대상 부위용 프라이머는 Primer3 프로그램을 이용하여 설계하였다.Mycobacterium tuberculosis complex (MTC: M. tuberculosis , M. bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe for detection of 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
(1) 결핵균(MTC)(1) Mycobacterium tuberculosis (MTC)
1) 대상 유전자: IS61101) Target gene: IS6110
2) 프라이머2) primer
a. 정방향 프라이머: 5′-cgaactcaaggagcacatca-3′(염기서열 1)a. Forward primer: 5'-cgaactcaaggagcacatca-3 '(SEQ ID NO: 1)
b. 역방향 프라이머: 5′-cagggttagccacactttgc-3′(염기서열 20)b. Reverse primer: 5′-cagggttagccacactttgc-3 ′ (SEQ ID NO: 20)
3) Taqman 프로브3) Taqman Probe
5′-HEX-cgccaactacggtgtttacggtg-BHQ1-3′(염기서열 21)5′-HEX-cgccaactacggtgtttacggtg-BHQ1-3 ′ (base sequence 21)
4) PCR 산물의 크기: 79bp4) PCR product size: 79bp
(2) 항산성비결핵균(NTM)(2) anti-acidic tuberculosis bacteria (NTM)
1) 대상유전자: 16S rRNA1) Target Gene: 16S rRNA
2) 프라이머2) primer
a. 정방향 프라이머: 5′-gtggcgaacgggtgagtaa-3′ (염기서열 22)a. Forward primer: 5′-gtggcgaacgggtgagtaa-3 ′ (SEQ ID NO: 22)
b. 역방향 프라이머b. Reverse primer
NTM-1: 5′-cccacaccgcaaaagctt-3′ (염기서열 5) 또는 5′-cccacaccgcaaaagct-3′ (염기서열 6) 또는 5′-tcccacaccgcaaaagct-3′ (염기서열 7)NTM-1: 5′-cccacaccgcaaaagctt-3 ′ (base 5) or 5′-cccacaccgcaaaagct-3 ′ (base 6) or 5′-tcccacaccgcaaaagct-3 ′ (base 7)
NTM-2: 5′-catcccacaccgctaccw-3′ (염기서열 8)NTM-2: 5′-catcccacaccgctaccw-3 ′ (SEQ ID NO: 8)
3) Taqman 프로브3) Taqman Probe
5′-FAM-cggtattagacccagtttcccaggct-BHQ1-3′(염기서열 23)5′-FAM-cggtattagacccagtttcccaggct-BHQ1-3 ′ (base sequence 23)
4) PCR 산물의 크기: 128bp4) Size of PCR product: 128bp
<실시예 2-1. 항산성비결핵균 검출용 역방향 프라이머 NTM-1으로 염기서열 5의 프라이머를 사용하는 이중 실시간 중합효소연쇄반응법><Example 2-1. Double real-time polymerase chain reaction using a primer of SEQ ID NO: 5 as a reverse primer NTM-1 for detecting acidic non-TB bacteria>
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 7주를 사용하였다. 사용된 ATCC 표준균주는 M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus (ATCC 19977), M. avium (ATCC 25291)이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated. The ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 7주를 사용하였다. 사용된 ATCC 표준균주는 M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus (ATCC 19977), M. avium (ATCC 25291)이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated. The ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC 표준균주는 액체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC standard strain was used by culturing in a liquid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)을 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 10초 간 변성, 약 65℃에서 약 15초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 2와 같다. 하기 primer-probes Mix에는 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있고 프루브는 4pmole/㎕이 있었다. 따라서 반응에 사용되는 MTC의 primer-probes mix 1.25㎕에는 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 프로브는 5pmole이 들어 있게 되었다. 총 25uL의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmoles/25㎕), 프로브는 0.2uM(5pmole/25㎕)가 사용되었다. NTM의 정방향 및 역방향 프라이머, 프로브의 농도 및 부피는 MTC와 동일하게 사용되었다. NTM-1 역방향 프라이머로는 염기서열 5의 프라이머가 NTM-2 역방향 프라이머로는 염기서열 8의 프라이머가 동량으로 사용하였다. NTM-2 역방향 프라이머는 5′-catcccacaccgctacct-3′ (염기서열 10)과 5′-catcccacaccgctacca-3′ (염기서열 11)이 동량으로 존재하도록 설계되었다.Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 10 seconds, and annealing and extension processes at about 65 ° C. for about 15 seconds. At this time, the composition of the reactants to perform the double real-time polymerase chain reaction is shown in Table 2. In the primer-probes Mix, the forward primer and the reverse primer were contained in the same amount (10 pmole / μl) and the probe had 4 pmole / μl. Therefore, 1.25 μl of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole. The total volume of the reactants for 25uL polymerization reaction was 25µl, so the concentration of primer was 0.5uM (12.5pmoles / 25µl) and the probe 0.2uM (5 pmole/25µl). The concentration and volume of forward and reverse primers, probes of NTM were used the same as MTC. As the NTM-1 reverse primer, the primer of base sequence 5 was used, and as the NTM-2 reverse primer, the primer of base sequence 8 was used in the same amount. NTM-2 reverse primers were designed such that 5′-catcccacaccgctacct-3 ′ (SEQ ID NO: 10) and 5′-catcccacaccgctacca-3 ′ (SEQ ID NO: 11) are present in equal amounts.
표 2
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
TABLE 2
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), VICTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . On the real-time monitor, it was designated to display on the green channel (510 ± 5nm) when the FAM TM was developed and on the yellow channel (555 ± 5nm) when the VIC TM was developed. Fluorescence was observed in the green channel and the yellow channel.
<실시예 2-2. 항산성비결핵균 검출용 역방향 프라이머 NTM-1으로 염기서열 6의 프라이머를 사용하는 이중 실시간 중합효소연쇄반응법><Example 2-2. Double real-time polymerase chain reaction using the primer of nucleotide sequence 6 as the reverse primer NTM-1 for the detection of non-acidic tuberculosis bacteria>
실시예 2-1과 NTM-1의 역방향 프라이머로 염기서열 6의 프라이머를 사용하는 것을 제외하고는 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Except for using the primer of SEQ ID NO: 6 as the reverse primer of Example 2-1 and NTM-1, the double real-time polymerase chain reaction was carried out in substantially the same manner.
<실시예 2-3. 항산성비결핵균 검출용 역방향 프라이머 NTM-1으로 염기서열 7의 프라이머를 사용하는 이중 실시간 중합효소연쇄반응법><Example 2-3. Double real-time polymerase chain reaction using primers of SEQ ID NO: 7 as reverse primer NTM-1
실시예 2-1과 NTM-1의 역방향 프라이머로 염기서열 7의 프라이머를 사용하는 것을 제외하고는 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Except for using the primer of SEQ ID NO: 7 as the reverse primer of Example 2-1 and NTM-1 was carried out a dual real-time polymerase chain reaction method in substantially the same manner.
2. 이중 실시간 중합효소연쇄반응법 수행 결과2. Result of double real time polymerase chain reaction
도 7 내지 도 12는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 7 및 도 8는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 노랑채널과 녹색채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 9 및 도 10는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 노랑채널과 녹색채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 11 및 도 12는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 노랑채널과 녹색채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.7 to 12 show the results obtained in the double real-time polymerase chain reaction method of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 7 and 8 are graphs showing the change in the fluorescence intensity of the cycle number of the polymerase chain reaction in the yellow channel and the green channel of the double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. 9 and 10 are graphs showing the change in fluorescence intensity for the number of cycles of the polymerase chain reaction in the yellow channel and the green channel of the double-real-time polymerase chain reaction of NRT. 11 and 12 show y-axis for the number of cycles of polymerase chain reaction in the yellow channel and the green channel of the double real-time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
도 7 내지 도 12를 참조하면, 결핵균(MTC)은 노랑채널에서 IS6110 유전자, 항산성비결핵균(NTM)은 녹색채널에서 16S rRNA 유전자, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 노랑채널과 녹색채널에서 IS6110 유전자와 16S rRNA 유전자가 특이적으로 증폭되는 결과를 나타내어, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.7 to 12, Mycobacterium tuberculosis (MTC) is an IS6110 gene in the yellow channel, acidic non-tuberculosis (NTM) is a 16S rRNA gene in the green channel, Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is a yellow channel and The results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel. When using the primers and probes according to the present invention, the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
실시예 3: 결핵균과 항산성비결핵균의 분리검출 방법 3Example 3: Separation and Detection Method of Mycobacterium Tuberculosis and Anti-acidic Tuberculosis
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
공통 프라이머로 마이코박테리아(mycobacteria)의 16S rRNA 유전자 부위를 증폭하고 결핵균(MTC: M. tuberculosis, M. bovis, M. africanum, M. microti)과 항산성비결핵균(NTM)을 특이적으로 구별할 수 있는 16S rRNA 염기부위를 Taqman 프로브로 사용하였다. 상기 검출대상 부위용 Taqman 프로브는 primer3 프로그램을 사용하여 설계하였다.Amplification of the 16S rRNA gene region of mycobacteria with consensus primers can be used to specifically distinguish Mycobacterium tuberculosis (MTC: M. tuberculosis , M. bovis , M. africanum , M. microti ) from acid-free tuberculosis (NTM) 16S rRNA base site was used as a Taqman probe. The detection site Taqman probe was designed using the primer3 program.
(1) 공통 프라이머(대상 유전자: 16S rRNA)(1) common primer (target gene: 16S rRNA)
a. 정방향 프라이머: 5′-ggataagcytgggaaactgg-3′ (염기서열 24)a. Forward primer: 5′-ggataagcytgggaaactgg-3 ′ (SEQ ID NO: 24)
b. 역방향 프라이머: 5′-accccaccaacwagctgata-3 ′(염기서열 25)b. Reverse primer: 5′-accccaccaacwagctgata-3 ′ (SEQ ID NO: 25)
(2) Taqman 프로브(대상 유전자: 16S rRNA)(2) Taqman probe (target gene: 16S rRNA)
a. 결핵균(MTC) Taqman 프로브a. Mycobacterium tuberculosis (MTC) Taqman probe
5′-VIC-tggtggaaagcgcttta-MGB-3′ (염기서열 26)5′-VIC-tggtggaaagcgcttta-MGB-3 ′ (SEQ ID NO: 26)
b. 항산성비결핵균 Taqman 프로브b. Antiacidic Mycobacterium Tuberculosis Taqman Probe
NTM-1: 5′-FAM-tggtggaaagcttttgc-MGB-3′ (염기서열 27)NTM-1: 5′-FAM-tggtggaaagcttttgc-MGB-3 ′ (SEQ ID NO: 27)
NTM-2: 5′-FAM-tggaaagygtttggtagc-MGB-3′ (염기서열 28)NTM-2: 5′-FAM-tggaaagygtttggtagc-MGB-3 ′ (SEQ ID NO: 28)
2. 이중 실시간 중합효소연쇄반응법2. Dual Real Time Polymerase Chain Reaction
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 7주를 사용하였다. 사용된 ATCC 표준균주는 M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus (ATCC 19977), M. avium (ATCC 25291)이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated. The ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 7주를 사용하였다. 사용된 ATCC 표준균주는 M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus (ATCC 19977), M. avium (ATCC 25291)이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic Mycobacterium tuberculosis, and 7 strains of Mycobacteria were isolated. The ATCC standard strains used were M. tuberculosis (ATCC 25177), M. intracellulare (ATCC 13950), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. fortuitum (ATCC 6841), M. abscessus ( ATCC 19977), M. avium (ATCC 25291).
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC 표준균주는 액체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC standard strain was used by culturing in a liquid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)을 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 15초 간 변성, 약 66℃에서 약 15초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 3과 같다. 하기 primer-probes Mix에는 마이코박테리아의 공통프라이머의 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있었고 MTC 프로브, NTM-1 프로브 및 NTM-2 프로브가 각각 4pmole/㎕씩 들어 있었다. 따라서, 반응에 사용되는 primer-probes mix 1.25㎕에는 공통 프라이머의 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 MTC, NTM-1, NTM-2 프로브가 각각 5pmole씩 들어있었다. 이에 따라, 총 25㎕의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmole/25㎕), MTC, NTM-1, NTM-2의 프로브는 0.2uM(5pmole/25㎕)의 농도를 가지도록 설계하였다. 이 때, 마이코박테리아의 염기서열 24의 정방향 프라이머는 5′-ggataagcctgggaaactgg-3′ (염기서열 29)과 5′-ggataagcttgggaaactgg-3′ (염기서열 30)이 동량인 6.25pmole씩 존재하도록 설계되었다. 또한, 염기서열 25의 역방향 프라이머는 5′-accccaccaacaagctgata-3′ (염기서열 31)과 5′-accccaccaactagctgata-3′ (염기서열 32)이 동량인 6.25pmole씩 존재하도록 설계되었다. 또한, NTM-2 프로브인 염기서열 28의 프로브는 5′-FAM-tggaaagcgtttggtagc-MGB-3′(염기서열 33)과 5′-FAM-tggaaagtgtttggtagc-MGB-3′ (염기서열 34)이 동량인 2.5pmole씩 존재하도록 설계되었다.Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were carried out using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 66 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 3. In the following primer-probes Mix, the forward primer and the reverse primer of the common primer of the mycobacteria were contained in the same amount (10 pmole / μl), and the MTC probe, NTM-1 probe and NTM-2 probe each contained 4 pmole / μl. Thus, 1.25 μl of primer-probes mix used for the reaction had forward and reverse primers of 12.5 pmole of common primer and 5 pmole of MTC, NTM-1, and NTM-2 probes, respectively. As a result, the total volume of the reaction product for performing the polymerization reaction of 25 μl was 25 μl. The concentration of the primer was 0.5 μM (12.5 pmole / 25 μl), and the probe of MTC, NTM-1, NTM-2 was 0.2 μM ( 5 pmole / 25 μl). At this time, the forward primers of the nucleotide sequence 24 of the mycobacteria were designed such that 5'-ggataagcctgggaaactgg-3 '(base sequence 29) and 5'-ggataagcttgggaaactgg-3' (base sequence 30) were present in the same amount of 6.25 pmole. In addition, the reverse primer of SEQ ID NO: 25 was designed such that 5'-accccaccaacaagctgata-3 '(base sequence 31) and 5'-accccaccaactagctgata-3' (base sequence 32) were present in equal amounts of 6.25 pmole. In addition, the probe of SEQ ID NO: 28, which is an NTM-2 probe, has the same amount of 5'-FAM-tggaaagcgtttggtagc-MGB-3 '(base sequence 33) and 5'-FAM-tggaaagtgtttggtagc-MGB-3' (base sequence 34). It is designed to exist by pmole.
표 3
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
TABLE 3
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), VICTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . On the real-time monitor, it was designated to display on the green channel (510 ± 5nm) when the FAM TM was developed and on the yellow channel (555 ± 5nm) when the VIC TM was developed. Fluorescence was observed in the green channel and the yellow channel.
3. 이중 실시간 중합효소연쇄반응법 수행 결과3. Result of Dual Real Time Polymerase Chain Reaction
도 13 내지 도 18는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 13 및 도 14는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 15 및 도 16는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 17 및 도 18는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.13 to 18 show the results obtained in the double real time polymerase chain reaction method of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 13 and 14 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. 15 and 16 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM). 17 and 18 are y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (MTC) + acidic non-TB tuberculosis (NTM), respectively, indicating a change in fluorescence intensity. It is a graph.
도 13 내지 도 18를 참조하면, 결핵균(MTC)은 노랑채널에서, 항산성비결핵균(NTM)은 녹색채널에서, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 녹색채널과 노랑채널에서 16S rRNA 유전자의 발현을 확인하여, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.Referring to Figures 13 to 18, Mycobacterium tuberculosis (MTC) in the yellow channel, acidic non-tuberculosis (NTM) in the green channel, Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is 16S rRNA in the green channel and yellow channel, respectively By confirming the expression of the gene, it was found that when using the primers and probes according to the present invention, it is possible to detect mycobacterium tuberculosis and anti-acidic non-tuberculosis bacterium at the same time with high reliability by the double real-time polymerase chain reaction method.
실시예 4: 결핵균과 항산성비결핵균의 분리검출 방법 4Example 4 Separation and Detection of Mycobacterium Tuberculosis and Antiacidic Tuberculosis
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
결핵균(Mycobacterium tuberculosis complex, MTC: M. tuberculosis, M.bovis, M. africanum, M. microti)은 IS6110 유전자를 검출 대상으로 하고, 항산성비결핵균(NTM)은 16S rRNA 유전자를 검출대상으로 하여 Taqman 프로브와 프라이머를 사용하였다. 상기 검출대상 부위용 프라이머는 Primer3 프로그램을 이용하여 설계하였다.Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe with 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
(1) 결핵균(MTC)(1) Mycobacterium tuberculosis (MTC)
1) 대상 유전자: IS61101) Target gene: IS6110
2) 프라이머2) primer
a. 정방향 프라이머: 5′-cgaactcaaggagcacatca-3′(염기서열 1)a. Forward primer: 5'-cgaactcaaggagcacatca-3 '(SEQ ID NO: 1)
b. 역방향 프라이머: 5′-cagggttagccacactttgc-3′(염기서열 20)b. Reverse primer: 5′-cagggttagccacactttgc-3 ′ (SEQ ID NO: 20)
3) Taqman 프로브3) Taqman Probe
5′-HEX-cgccaactacggtgtttacggtg-BHQ1-3′(염기서열 21)5′-HEX-cgccaactacggtgtttacggtg-BHQ1-3 ′ (base sequence 21)
4) PCR 산물의 크기: 79bp4) PCR product size: 79bp
(2) 항산성비결핵균(NTM)(2) anti-acidic tuberculosis bacteria (NTM)
1) 대상유전자: 16S rRNA1) Target Gene: 16S rRNA
2) 프라이머2) primer
a. 정방향 프라이머: 5′-catgtyttstggkgsaaagctt-3′ (염기서열 35)a. Forward primer: 5′-catgtyttstggkgsaaagctt-3 ′ (SEQ ID NO: 35)
b. 역방향 프라이머: 5′-cgtaggagtctgggccgta-3′ (염기서열 36)b. Reverse primer: 5′-cgtaggagtctgggccgta-3 ′ (SEQ ID NO: 36)
3) Taqman 프로브3) Taqman Probe
FAM-tagccggcctgagagggtg-BHQ1 (염기서열 37) 또는 FAM-cctgagagggtgwccggcc-BHQ1 (염기서열 38) 또는 FAM-cgggtagccggcctgagag-BHQ1 (염기서열 39)FAM-tagccggcctgagagggtg-BHQ1 (base sequence 37) or FAM-cctgagagggtgwccggcc-BHQ1 (base sequence 38) or FAM-cgggtagccggcctgagag-BHQ1 (base sequence 39)
4) PCR 산물의 크기: 152bp4) Size of PCR product: 152bp
<실시예 4-1. 항산성비결핵균 검출용 프로브로 염기서열 37의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 4-1. Dual Real-Time Polymerase Chain Reaction Using Taqman Probe of SEQ ID NO: 37 as a Probe for the Detection of Non-acidic Mycobacterium Tuberculosis>
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 68주를 사용하였다. 사용된 표준균주는 M. abscessus ATCC 19977, M. acapulcensis KCTC 9501, M. africanum ATCC 25420, M. agri KCTC 9502, M. alvei KCTC 19709, M. asiaticum KCTC 9503, M. aurum KCTC 19457, M. austroafricanum KCTC 9504, M. avium ATCC 25291, M. bolletii KCTC 19281, M. botniense KCTC 19646, M. bovis ATCC 19210, M. brumae KCTC 19711, M. celatum ATCC 51131, M. chelonae subsp chelonae KCTC 9505, M. chlorophenolicum KCTC 19089, M. chubuense KCTC 19712, M. diernhoferi KCTC 9506, M. fallax KCTC 9508, M. flavescens ATCC 14474, M. fortuitum ATCC 6841, M. frederiksbergense KCTC 19100, M. gadium ATCC 27726, M. gastri ATCC 15754, M. gilvum KCTC 19423, M. goodii ATCC BAA-955, M. gordonae KCTC 9513, M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950, M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571, M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M. neoaurum KCTC 19096, M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979, M. peregrinum KCTC 9615, KMRC 75002, M. phlei KCTC 9689, M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108, M. szulgai KCTC 9520, KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M. vanbaalenii KCTC 9966, M. wolinskyi ATCC 700010, M. xenopi KMRC 42001이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic tuberculosis, and 68 strains of Mycobacteria were isolated from clinical specimens. Standard strains used were M. abscessus ATCC 19977 , M. acapulcensis KCTC 9501, M. africanum ATCC 25420, M. agri KCTC 9502, M. alvei KCTC 19709, M. asiaticum KCTC 9503, M. aurum KCTC 19457, M. austroafricanum KCTC 9504, M. avium ATCC 25291, M. bolletii KCTC 19281, M. botniense KCTC 19646, M. bovis ATCC 19210, M. brumae KCTC 19711, M. celatum ATCC 51131, M. chelonae subsp chelonae KCTC 9505, M. chlorophenolicum KCTC 19089, M. chubuense KCTC 19712, M. diernhoferi KCTC 9506, M. fallax KCTC 9508, M. flavescens ATCC 14474, M. fortuitum ATCC 6841, M. frederiksbergense KCTC 19100, M. gadium ATCC 27726, M. gastri ATCC 15754 , M. gilvum KCTC 19423, M. goodii ATCC BAA-955, M. gordonae KCTC 9513, M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950 , M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571 , M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M. neoaurum KCTC 19096, M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979 , M. peregrinum KCTC 9615, KMRC 75002, M. phlei KCTC 9689 , M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108 , M. szulgai KCTC 9520, KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M vanbaalenii KCTC 9966 , M. wolinskyi ATCC 700010, M. xenopi KMRC 42001.
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC, KCTC 균주는 액체배지에서 배양하여 사용하였고, KMRC 균주는 고체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)을 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 12초 간 변성, 약 63℃에서 약 12초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 4와 같다. 하기 primer-probes Mix에는 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있고 프루브는 4pmole/㎕이 있었다. 따라서 반응에 사용되는 MTC의 primer-probes mix 1.25㎕에는 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 프로브는 5pmole이 들어 있게 되었다. 총 25㎕의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmoles/25㎕), 프로브는 0.2uM(5pmole/25㎕)가 사용되었다. NTM의 정방향 및 역방향 프라이머, 프로브의 농도는 MTC와 동일하게 사용되었다. 이 때, NTM 정방향 프라이머(염기서열 35)는 5′-catgtcttgtgggggaaagctt-3′ (염기서열 40), 5′-catgttttgtgggggaaagctt-3′ (염기서열 41), 5′-catgtcttctgggggaaagctt-3′ (염기서열 42), 5′-catgtcttgtggtggaaagctt-3′ (염기서열 43), 5′-catgtcttgtggggcaaagctt-3′ (염기서열 44), 5′-catgttttctgggggaaagctt-3′ (염기서열 45), 5′-catgtcttctggtggaaagctt-3′ (염기서열 46), 5′-catgtcttgtggtgcaaagctt-3′ (염기서열 47), 5′-catgttttgtggggcaaagctt-3′ (염기서열 48), 5′-catgtcttctggggcaaagctt-3′ (염기서열 49), 5′-catgttttgtggtggaaagctt-3′ (염기서열 50), 5′-catgttttctggtggaaagctt-3′ (염기서열 51), 5′-catgttttctggggcaaagctt-3′ (염기서열 52), 5′-catgttttgtggtgcaaagctt-3′ (염기서열 53), 5′-catgtcttctggtgcaaagctt-3′ (염기서열 54) 및 5′-catgttttctggtgcaaagctt-3′ (염기서열 55)이 약 1:1:1:1:1:1:1:1:1:1:1:1:1:1:1:1로 동량으로 존재하였다.Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 12 seconds, and annealing and extension process at about 63 ° C. for about 12 seconds. At this time, the composition of the reactants performing the double real-time polymerase chain reaction is shown in Table 4. In the following primer-probes Mix, the forward primer and the reverse primer contained the same amount (10 pmole / μl) and the probe was 4 pmole / μl. Therefore, 1.25 μl of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole. The total volume of the reaction product to perform 25 μL of polymerization chain reaction was 25 μL. The concentration of the primer was 0.5 μM (12.5 pmoles / 25 μL) and the probe was 0.2 μM (5 pmole / 25 μL). The concentrations of the forward and reverse primers and probes of NTM were used the same as MTC. NTM forward primer (base sequence 35) is 5′-catgtcttgtgggggaaagctt-3 ′ (base sequence 40), 5′-catgttttgtgggggaaagctt-3 ′ (base sequence 41), 5′-catgtcttctgggggaaagctt-3 ′ (base sequence 42) , 5′-catgtcttgtggtggaaagctt-3 ′ (base 43), 5′-catgtcttgtggggcaaagctt-3 ′ (base 44), 5′-catgttttctgggggaaagctt-3 ′ (base 45), 5′-catgtcttctggtggaaagctt-3 ′ (base sequence) 46), 5′-catgtcttgtggtgcaaagctt-3 ′ (base sequence 47), 5′-catgttttgtggggcaaagctt-3 ′ (base sequence 48), 5′-catgtcttctggggcaaagctt-3 ′ (base sequence 49), 5′-catgttttgtggtggaaagctt-3 ′ (baseline) SEQ ID NO: 50), 5′-catgttttctggtggaaagctt-3 ′ (SEQ ID NO: 51), 5′-catgttttctggggcaaagctt-3 ′ (SEQ ID NO: 52), 5′-catgttttgtggtgcaaagctt-3 ′ (SEQ ID NO: 53), 5′-catgtcttctggtgcaaagctt-3 ′ (Base sequence 54) and 5′-catgttttctggtgcaaagctt-3 ′ (base sequence 55) are about 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1 It was present in the same amount as 1: 1.
표 4
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
Table 4
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), HexTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . On the real-time monitor, it was designated to display the green channel (510 ± 5nm) when the FAM TM was developed and the yellow channel (555 ± 5 nm) when the Hex TM was developed. Fluorescence was observed in the green channel and the yellow channel.
<실시예 4-2. 항산성비결핵균 검출용 프로브로 염기서열 38의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 4-2. Double Real-Time Polymerase Chain Reaction Using Taqman Probe of Base Sequence 38
실시예 4-1과 항산성비결핵균 검출용 프로브로 염기서열 38의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다. 염기서열 38의 프로브는 FAM-cctgagagggtgaccggcc-BHQ1 (염기서열 56) 및 FAM-cctgagagggtgtccggcc-BHQ1 (염기서열 57)이 동량으로 존재하도록 설계된 것이다.Except for using the Taqman probe of SEQ ID NO: 38 as Example 4-1 and the anti-acidic tuberculosis bacteria detection probe was carried out in a double real-time polymerase chain reaction method in substantially the same manner. The probe of SEQ ID NO: 38 is designed so that FAM-cctgagagggtgaccggcc-BHQ1 (base sequence 56) and FAM-cctgagagggtgtccggcc-BHQ1 (base sequence 57) are present in equal amounts.
<실시예 4-3. 항산성비결핵균 검출용 프로브로 염기서열 39의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 4-3. Double Real-Time Polymerase Chain Reaction Using Taqman Probe of SEQ ID NO: 39 as a Probe for Detection of Non-acidic Mycobacterium Tuberculosis>
실시예 4-1과 항산성비결핵균 검출용 프로브로 염기서열 39의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Except for using the Taqman probe of SEQ ID NO: 39 as Example 4-1 and the anti-acidic tuberculosis bacteria detection probe was carried out in a double real-time polymerase chain reaction method in substantially the same manner.
2. 이중 실시간 중합효소연쇄반응법 수행 결과2. Result of double real time polymerase chain reaction
도 19 내지 도 24는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 19 및 도 20는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 21 및 도 22는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 23 및 도 24는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.19 to 24 show the results obtained in the double real-time polymerase chain reaction method of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 19 and 20 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. 21 and 22 are graphs showing the change in fluorescence intensity of the y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction (NTM). FIG. 23 and FIG. 24 show y-axis for the number of cycles of polymerase chain reaction in green and yellow channels of dual real time polymerase chain reaction (MTC) + antiacidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
도 19 내지 도 24를 참조하면, 결핵균(MTC)은 노랑채널에서 IS6110 유전자, 항산성비결핵균(NTM)은 녹색채널에서 16S rRNA 유전자, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 노랑채널과 녹색채널에서 IS6110 유전자와 16S rRNA 유전자가 특이적으로 증폭되는 결과를 나타내어, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.19 to 24, the tuberculosis bacterium (MTC) is an IS6110 gene in the yellow channel, the acidic non-tuberculosis bacterium (NTM) is the 16S rRNA gene in the green channel, Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is the yellow channel and The results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel. When using the primers and probes according to the present invention, the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
실시예 5: 결핵균과 항산성비결핵균의 분리검출 방법 5Example 5 Separation and Detection Method of Mycobacterium Tuberculosis and Antiacidic Tuberculosis
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
결핵균(Mycobacterium tuberculosis complex, MTC: M. tuberculosis, M.bovis, M. africanum, M. microti)은 IS6110 유전자를 검출 대상으로 하고, 항산성비결핵균(NTM)은 16S rRNA 유전자를 검출대상으로 하여 Taqman 프로브와 프라이머를 사용하였다. 상기 검출대상 부위용 프라이머는 Primer3 프로그램을 이용하여 설계하였다. Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe for detection of 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
(1) 결핵균(MTC)(1) Mycobacterium tuberculosis (MTC)
1) 대상 유전자: IS61101) Target gene: IS6110
2) 프라이머2) primer
a. 정방향 프라이머: 5′-cgaactcaaggagcacatcag-3′(염기서열 58)a. Forward primer: 5'-cgaactcaaggagcacatcag-3 '(SEQ ID NO: 58)
b. 역방향 프라이머: 5′-gagtttggtcatcagccgttc-3′(염기서열 59)b. Reverse primer: 5′-gagtttggtcatcagccgttc-3 ′ (SEQ ID NO: 59)
3) Taqman 프로브3) Taqman Probe
5′-HEX-cgccaactacggtgtttacggtg-BHQ1-3′(염기서열 21) 또는5′-HEX-cgccaactacggtgtttacggtg-BHQ1-3 ′ (base sequence 21) or
5′-VIC-agtgtggctaaccctgaac-MGB-3′(염기서열 60)5′-VIC-agtgtggctaaccctgaac-MGB-3 ′ (base sequence 60)
4) PCR 산물의 크기: 136bp4) Size of PCR product: 136bp
(2) 항산성비결핵균(NTM)(2) anti-acidic tuberculosis bacteria (NTM)
1) 대상유전자: 16S rRNA1) Target Gene: 16S rRNA
2) 프라이머2) primer
a. 정방향 프라이머: 5′-tggcgaacgggtgagtaa-3′ (염기서열 61)a. Forward primer: 5′-tggcgaacgggtgagtaa-3 ′ (SEQ ID NO: 61)
b. 역방향 프라이머b. Reverse primer
5′-cccacaccgcaaaagctt-3′ (염기서열 5)5′-cccacaccgcaaaagctt-3 ′ (SEQ ID NO: 5)
5′-catcccacaccgctaccw-3′ (염기서열 8)5′-catcccacaccgctaccw-3 ′ (SEQ ID NO: 8)
3) Taqman 프로브3) Taqman Probe
5′-FAM-cggtattagacccagtttcccagg-BHQ1-3′(염기서열 62) 또는 5′-FAM-cggtattagacccagtttcccagg-BHQ1-3 ′ (base sequence 62) or
5′-FAM-tgggaaactgggtctaatac-MGB-3′(염기서열 63)5′-FAM-tgggaaactgggtctaatac-MGB-3 ′ (base sequence 63)
4) PCR 산물의 크기: 127bp4) Size of PCR product: 127bp
<실시예 5-1. 결핵균(MTC) 검출용 프로브로 염기서열 21의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 62의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 5-1. Dual real-time polymerase chain reaction using Taqman probe of SEQ ID NO: 21 as a probe for detecting Mycobacterium tuberculosis (MTC) and Taqman probe of SEQ ID NO: 62 as a probe for detecting non-acidic Mycobacterium tuberculosis>
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 68주를 사용하였다. 사용된 표준균주는 M. abscessus ATCC 19977, M. acapulcensis KCTC 9501, M. africanum ATCC 25420, M. agri KCTC 9502, M. alvei KCTC 19709, M. asiaticum KCTC 9503, M. aurum KCTC 19457, M. austroafricanum KCTC 9504, M. avium ATCC 25291, M. bolletii KCTC 19281, M. botniense KCTC 19646, M. bovis ATCC 19210, M. brumae KCTC 19711, M. celatum ATCC 51131, M. chelonae subsp chelonae KCTC 9505, M. chlorophenolicum KCTC 19089, M. chubuense KCTC 19712, M. diernhoferi KCTC 9506, M. fallax KCTC 9508, M. flavescens ATCC 14474, M. fortuitum ATCC 6841, M. frederiksbergense KCTC 19100, M. gadium ATCC 27726, M. gastri ATCC 15754, M. gilvum KCTC 19423, M. goodii ATCC BAA-955, M. gordonae KCTC 9513, M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950, M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571, M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M. neoaurum KCTC 19096, M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979, M. peregrinum KCTC 9615, KMRC 75002, M. phlei KCTC 9689, M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108, M. szulgai KCTC 9520, KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M. vanbaalenii KCTC 9966, M. wolinskyi ATCC 700010, M. xenopi KMRC 42001이었다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic tuberculosis, and 68 strains of Mycobacteria were isolated from clinical specimens. Standard strains used were M. abscessus ATCC 19977 , M. acapulcensis KCTC 9501, M. africanum ATCC 25420, M. agri KCTC 9502, M. alvei KCTC 19709, M. asiaticum KCTC 9503, M. aurum KCTC 19457, M. austroafricanum KCTC 9504, M. avium ATCC 25291, M. bolletii KCTC 19281, M. botniense KCTC 19646, M. bovis ATCC 19210, M. brumae KCTC 19711, M. celatum ATCC 51131, M. chelonae subsp chelonae KCTC 9505, M. chlorophenolicum KCTC 19089, M. chubuense KCTC 19712, M. diernhoferi KCTC 9506, M. fallax KCTC 9508, M. flavescens ATCC 14474, M. fortuitum ATCC 6841, M. frederiksbergense KCTC 19100, M. gadium ATCC 27726, M. gastri ATCC 15754 , M. gilvum KCTC 19423, M. goodii ATCC BAA-955, M. gordonae KCTC 9513, M. haemophilum ATCC 29548, M. hassiacum ATCC 700660, M. interjectum ATCC 51457, M. intermedium ATCC 51848, M. intracellulare ATCC 13950 , M. intracellulare KCTC 9514, M. kansasii ATCC 12478, M. lentiflavum KMRC 70087, M. malmoense ATCC 29571 , M. mantobense KCTC 9977, M. marinum ATCC 927, M. massiliense KCTC 19086, M. microti ATCC 19422, M. moriokaense KCTC 9516, M. mucogenicum KCTC 19088, M. neoaurum KCTC 19096, M. nonchromogenicum ATCC 19530, M. obuense KCTC 19097, M. parascrofulaceum KCTC 9979 , M. peregrinum KCTC 9615, KMRC 75002, M. phlei KCTC 9689 , M. porcinum KCTC 9517, M. pulveris KCTC 9518, M. scrofulaceum ATCC 19981, M. septicum ATCC 700731, M. simiae ATCC 25275, M. shimoidei ATCC 27962, M. smegmatis KCTC 9108 , M. szulgai KCTC 9520, KMRC 31125, M. terrae KCTC 9614, M. triplex ATCC 700071, M. triviale KMRC 70093, M. tuberculosis ATCC 25177, ATCC 27294, M. ulcerans ATCC 19423, M. vaccae KCTC 19087, M vanbaalenii KCTC 9966 , M. wolinskyi ATCC 700010, M. xenopi KMRC 42001.
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC, KCTC 균주는 액체배지에서 배양하여 사용하였고, KMRC 균주는 고체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)을 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 15초 간 변성, 약 65℃에서 약 15초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 5와 같다. 하기 primer-probes Mix에는 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있고 프루브는 4pmole/㎕이 있었다. 따라서 반응에 사용되는 MTC의 primer-probes mix 1.25㎕에는 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 프로브는 5pmole이 들어 있게 되었다. 총 25uL의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmoles/25㎕), 프로브는 0.2uM(5pmole/25㎕)가 사용되었다. NTM의 정방향 및 역방향 프라이머, 프로브의 농도는 MTC와 동일하게 사용되었다. Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 65 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 5. In the following primer-probes Mix, the forward primer and the reverse primer contained the same amount (10 pmole / μl) and the probe was 4 pmole / μl. Therefore, 1.25 μl of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole. The total volume of 25uL of polymerase chain reaction was 25µL. The primer concentration was 0.5uM (12.5pmoles / 25µl) and the probe 0.2uM (5 pmole/25µl). The concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
표 5
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
Table 5
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), HexTM나 VICTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . It is designated to display in the green channel (510 ± 5nm) when the FAM TM is developed on the real time monitor, and in the yellow channel (555 ± 5nm) when the Hex TM or VIC TM is developed. Fluorescence was observed in the green channel and the yellow channel.
<실시예 5-2. 결핵균(MTC) 검출용 프로브로 염기서열 21의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 63의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 5-2. Dual real-time polymerase chain reaction using Taqman probe of SEQ ID NO: 21 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of SEQ ID NO: 63 as a probe for detecting acidic non-TB bacteria>
실시예 5-1과 결핵균(MTC) 검출용 프로브로 염기서열 21의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 63의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Dual real-time in substantially the same manner as Example 5-1 and Taqman probe of SEQ ID NO: 21 as the probe for detecting Mycobacterium tuberculosis (MTC), and Taqman probe of SEQ ID NO: 63 as the probe for detecting the non-acidic Mycobacterium tuberculosis bacterium Polymerase chain reaction was performed.
<실시예 5-3. 결핵균(MTC) 검출용 프로브로 염기서열 60의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 62의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 5-3. Dual real-time polymerase chain reaction using Taqman probe of base sequence 60 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base sequence 62 as a probe for detecting non-malignant tuberculosis bacteria>
실시예 5-1과 결핵균(MTC) 검출용 프로브로 염기서열 60의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 62의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Dual real-time in the same manner as in Example 5-1 except that a Taqman probe of SEQ ID NO: 60 was used as a probe for detection of Mycobacterium tuberculosis (MTC), and a Taqman probe of SEQ ID NO: 62 was used as a probe for detecting an acidic non-TB bacterium. Polymerase chain reaction was performed.
<실시예 5-4. 결핵균(MTC) 검출용 프로브로 염기서열 60의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 63의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 5-4. Dual real-time polymerase chain reaction using Taqman probe of base sequence 60 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base sequence 63 as a probe for detecting non-malignant tuberculosis bacteria>
실시예 5-1과 결핵균(MTC) 검출용 프로브로 염기서열 60의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 63의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Dual real-time in substantially the same manner as in Example 5-1 and using the Taqman probe of SEQ ID NO: 60 as the probe for detecting Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 63 as the probe for the detection of non-acidic tuberculosis bacteria Polymerase chain reaction was performed.
2. 이중 실시간 중합효소연쇄반응법 수행 결과2. Result of double real time polymerase chain reaction
도 25 내지 도 30는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 25 및 도 26는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 27 및 도 28는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 29 및 도 30는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.25 to 30 show the results obtained in the double real-time polymerase chain reaction method of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 25 and 26 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. FIG. 27 and FIG. 28 are graphs showing changes in fluorescence intensity of the cycle number of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction of NRT. 29 and 30 show y-axis for the number of cycles of the polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
도 25 내지 도 30를 참조하면, 결핵균(MTC)은 노랑채널에서 IS6110 유전자, 항산성비결핵균(NTM)은 녹색채널에서 16S rRNA 유전자, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 노랑채널과 녹색채널에서 IS6110 유전자와 16S rRNA 유전자가 특이적으로 증폭되는 결과를 나타내어, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.25 to 30, Mycobacterium tuberculosis (MTC) is the IS6110 gene in the yellow channel, acidic non-tuberculosis (NTM) is the 16S rRNA gene in the green channel, Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is the yellow channel and The results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel. When using the primers and probes according to the present invention, the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
실시예 6: 결핵균과 항산성비결핵균의 분리검출 방법 6Example 6: Separation and Detection Method of Mycobacterium Tuberculosis and Antiacidic Tuberculosis
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
결핵균(Mycobacterium tuberculosis complex, MTC: M. tuberculosis, M.bovis, M. africanum, M. microti)은 IS6110 유전자를 검출 대상으로 하고, 항산성비결핵균(NTM)은 16S rRNA 유전자를 검출대상으로 하여 Taqman 프로브와 프라이머를 사용하였다. 상기 검출대상 부위용 프라이머는 Primer3 프로그램을 이용하여 설계하였다. Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB is a Taqman probe for detection of 16S rRNA gene. And primers were used. The primer for the detection site was designed using the Primer3 program.
(1) 결핵균(Mycobacterium tuberculosis complex, MTC)(1) Mycobacterium tuberculosis complex (MTC)
1) 대상 유전자: IS61101) Target gene: IS6110
2) 프라이머2) primer
a. 정방향 프라이머: 5′-cgaactcaaggagcacatcag-3′(염기서열 58)a. Forward primer: 5'-cgaactcaaggagcacatcag-3 '(SEQ ID NO: 58)
b. 역방향 프라이머: 5′-cagggttagccacactttgc-3′(염기서열 20)b. Reverse primer: 5′-cagggttagccacactttgc-3 ′ (SEQ ID NO: 20)
3) Taqman 프로브3) Taqman Probe
5′-Hex-cgccaactacggtgtttacggtg-BHQ1-3′(염기서열 21) 또는5′-Hex-cgccaactacggtgtttacggtg-BHQ1-3 ′ (base sequence 21) or
5′-VIC-ctacggtgtttacggtgc-MGB-3′(염기서열 64)5′-VIC-ctacggtgtttacggtgc-MGB-3 ′ (base sequence 64)
4) PCR 산물의 크기: 79bp4) PCR product size: 79bp
(2) 항산성비결핵균(Nontuberculous mycobacteria, NTM)(2) Nontuberculous mycobacteria (NTM)
1) 대상유전자: 16S rRNA1) Target Gene: 16S rRNA
2) 프라이머2) primer
a. 정방향 프라이머 a. Forward primer
NTM-1: 5′-tktggtggaaagcttttgc-3′(염기서열 65)NTM-1: 5′-tktggtggaaagcttttgc-3 ′ (SEQ ID NO: 65)
NTM-2: 5′-ggtgwgtggtgcaaagctt-3′(염기서열 66)NTM-2: 5′-ggtgwgtggtgcaaagctt-3 ′ (SEQ ID NO: 66)
NTM-3: 5′-tggtggaaagcgtttggt-3′(염기서열 67)NTM-3: 5′-tggtggaaagcgtttggt-3 ′ (SEQ ID NO: 67)
b. 역방향 프라이머: 5′-cgtaggagtctgggccgta-3′ (염기서열 36)b. Reverse primer: 5′-cgtaggagtctgggccgta-3 ′ (SEQ ID NO: 36)
3) Taqman 프로브 3) Taqman Probe
5′-FAM-cgggtagccggcctgagag-BHQ1-3′(염기서열 39) 또는5′-FAM-cgggtagccggcctgagag-BHQ1-3 ′ (base sequence 39) or
5′-FAM-cctgagagggtgwccg-MGB-3′(염기서열 68)5′-FAM-cctgagagggtgwccg-MGB-3 ′ (base sequence 68)
4) PCR 산물의 크기: 146bp ~ 148bp4) PCR product size: 146bp ~ 148bp
<실시예 6-1. 결핵균(MTC) 검출용 프로브로 염기서열 21의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 39의 Taqman 프로브를 사용하는 이중 중합효소연쇄반응법><Example 6-1. Double polymerase chain reaction using Taqman probe of base sequence 21 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base sequence 39 as a probe for detecting non-malignant tuberculosis bacteria>
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 68주를 사용하였다. 사용된 표준균주는 실시예 5-1에서 언급한 것과 실질적으로 동일하다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic tuberculosis strain, and 68 strains of Mycobacteria were isolated from clinical specimens. The standard strain used was substantially the same as mentioned in Example 5-1.
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC, KCTC 균주는 액체배지에서 배양하여 사용하였고, KMRC 균주는 고체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)을 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 15초 간 변성, 약 64℃에서 약 15초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 6과 같다. 하기 primer-probes Mix에는 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있고 프루브는 4pmole/㎕이 있었다. 따라서 반응에 사용되는 MTC의 primer-probes mix 1.25㎕에는 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 프로브는 5pmole이 들어 있게 되었다. 총 25uL의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmoles/25㎕), 프로브는 0.2uM(5pmole/25㎕)가 사용되었다. NTM의 정방향 및 역방향 프라이머, 프로브의 농도는 MTC와 동일하게 사용되었다. Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 64 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 6. In the following primer-probes Mix, the forward primer and the reverse primer contained the same amount (10 pmole / μl) and the probe was 4 pmole / μl. Therefore, 1.25 μl of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole. The total volume of 25uL of polymerase chain reaction was 25µL. The primer concentration was 0.5uM (12.5pmoles / 25µl) and the probe 0.2uM (5 pmole/25µl). The concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
표 6
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
Table 6
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), HexTM나 VICTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . It is designated to display in the green channel (510 ± 5nm) when the FAM TM is developed on the real time monitor, and in the yellow channel (555 ± 5nm) when the Hex TM or VIC TM is developed. Fluorescence was observed in the green channel and the yellow channel.
<실시예 6-2. 결핵균(MTC) 검출용 프로브로 염기서열 21의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 68의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 6-2. Dual real-time polymerase chain reaction using Taqman probe of SEQ ID NO: 21 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of SEQ ID NO: 68 as a probe for detecting acidic non-TB bacteria>
실시예 6-1과 결핵균(MTC) 검출용 프로브로 염기서열 21의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 68의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Dual real-time in substantially the same manner as in Example 6-1 except that the Taqman probe of SEQ ID NO: 21 was used as a probe for the detection of Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 68 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
<실시예 6-3. 결핵균(MTC) 검출용 프로브로 염기서열 64의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 39의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 6-3. Dual real-time polymerase chain reaction using Taqman probe of base sequence 64 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base sequence 39 as a probe for detecting non-malignant tuberculosis bacteria>
실시예 6-1과 결핵균(MTC) 검출용 프로브로 염기서열 64의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 39의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Dual real-time in substantially the same manner as in Example 6-1, except that the Taqman probe of SEQ ID NO: 64 was used as a probe for the detection of Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 39 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
<실시예 6-4. 결핵균(MTC) 검출용 프로브로 염기서열 64의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 68의 Taqman 프로브를 사용하는 이중 실시간 중합효소연쇄반응법><Example 6-4. Dual real-time polymerase chain reaction using Taqman probe of base sequence 64 as a probe for detection of Mycobacterium tuberculosis (MTC) and Taqman probe of base 68 as a probe for detecting non-malignant tuberculosis bacteria>
실시예 6-1과 결핵균(MTC) 검출용 프로브로 염기서열 64의 Taqman 프로브를 사용하고, 항산성비결핵균 검출용 프로브로 염기서열 68의 Taqman 프로브를 사용하는 것을 제외하고 실질적으로 동일한 방법으로 이중 실시간 중합효소연쇄반응법을 수행하였다.Dual real-time in the same manner as in Example 6-1 except that the Taqman probe of SEQ ID NO: 64 was used as a probe for the detection of Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 68 was used as the probe for the detection of non-malignant tuberculosis bacteria. Polymerase chain reaction was performed.
2. 이중 실시간 중합효소연쇄반응법 수행 결과2. Result of double real time polymerase chain reaction
도 31 내지 도 36는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 31 및 도 32는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 33 및 도 34는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 35 및 도 36는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.31 to 36 show the results obtained in the double real time polymerase chain reaction method of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 31 and 32 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reactions in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. 33 and 34 are graphs showing changes in fluorescence intensity of cycles of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction (NTM). 35 and 36 show the y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively, in the green channel and the yellow channel. It is a graph.
도 31 내지 도 36를 참조하면, 결핵균(MTC)은 노랑채널에서 IS6110 유전자, 항산성비결핵균(NTM)은 녹색채널에서 16S rRNA 유전자, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 노랑채널과 녹색채널에서 IS6110 유전자와 16S rRNA 유전자가 특이적으로 증폭되는 결과를 나타내어, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.31 to 36, Mycobacterium tuberculosis (MTC) is the IS6110 gene in the yellow channel, acidic non-tuberculosis (NTM) is the 16S rRNA gene in the green channel, Mycobacterium tuberculosis (MTC) + acidic non-tuberculosis (NTM) is the yellow channel and The results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel. When using the primers and probes according to the present invention, the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
실시예 7: 결핵균과 항산성비결핵균의 분리검출 방법 7Example 7 Separation and Detection Method of Mycobacterium Tuberculosis and Anti-acidic Tuberculosis
1. 검출대상 부위 및 프라이머 설계1. Detection site and primer design
결핵균(Mycobacterium tuberculosis complex, MTC: M. tuberculosis, M.bovis, M. africanum, M. microti)은 IS6110 유전자를 검출 대상으로 하고, 항산성비결핵균(NTM)은 16S rRNA 유전자를 특이적으로 구별할 수 있는 16S rRNA 염기부위를 Taqman 프로브로 사용하였다. 16S rRNA 유전자의 경우 공통프라이머로 마이코박테아의 16S rRNA 유전자 부위를 증폭할 수 있는 프라이머를 사용하였다. 상기 검출대상 부위용 프라이머는 Primer3 프로그램을 이용하여 설계하였다. Mycobacterium tuberculosis complex (MTC: M. tuberculosis, M.bovis , M. africanum , M. microti ) is an IS6110 gene for detection, and NTB can specifically distinguish 16S rRNA genes. 16S rRNA base site was used as a Taqman probe. In the case of 16S rRNA gene, a primer capable of amplifying the 16S rRNA gene region of Mycobactera was used as a common primer. The primer for the detection site was designed using the Primer3 program.
(1) 결핵균(Mycobacterium tuberculosis complex, MTC)(1) Mycobacterium tuberculosis complex (MTC)
1) 대상 유전자: IS61101) Target gene: IS6110
2) 프라이머2) primer
a. 정방향 프라이머: 5′-cgaactcaaggagcacatcag-3′(염기서열 58)a. Forward primer: 5'-cgaactcaaggagcacatcag-3 '(SEQ ID NO: 58)
b. 역방향 프라이머: 5′-gagtttggtcatcagccgttc-3′(염기서열 59)b. Reverse primer: 5′-gagtttggtcatcagccgttc-3 ′ (SEQ ID NO: 59)
3) Taqman 프로브3) Taqman Probe
5′-VIC-agtgtggctaaccctgaac-MGB-3′(염기서열 60)5′-VIC-agtgtggctaaccctgaac-MGB-3 ′ (base sequence 60)
4) PCR 산물의 크기: 136bp4) Size of PCR product: 136bp
(2) 항산성비결핵균(Nontuberculous mycobacteria, NTM)(2) Nontuberculous mycobacteria (NTM)
1) 대상유전자: 16S rRNA1) Target Gene: 16S rRNA
2) 프라이머2) primer
a. 정방향 프라이머: 5′-ggataagcytgggaaactgg-3′(염기서열 24)a. Forward primer: 5′-ggataagcytgggaaactgg-3 ′ (SEQ ID NO: 24)
b. 역방향 프라이머: 5′-cgtaggagtctgggccgta-3′(염기서열 75)b. Reverse primer: 5′-cgtaggagtctgggccgta-3 ′ (base sequence 75)
3) Taqman 프로브 3) Taqman Probe
NTM-1: 5′-FAM-tggtggaaagcttttgc-MGB-3′(염기서열 27)NTM-1: 5′-FAM-tggtggaaagcttttgc-MGB-3 ′ (SEQ ID NO: 27)
NTM-2: 5′-FAM-ccacaccgctaccaaac-MGB-3′(염기서열 76)NTM-2: 5′-FAM-ccacaccgctaccaaac-MGB-3 ′ (SEQ ID NO: 76)
4) PCR 산물의 크기: 205bp4) Size of PCR product: 205bp
2. 이중 실시간 중합효소연쇄반응법2. Dual Real Time Polymerase Chain Reaction
(1) DNA의 분리(1) Isolation of DNA
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주, 마이코박테리아 표준 균주 68주를 사용하였다. 사용된 표준균주는 실시예 5-1에서 언급한 것과 실질적으로 동일하다.186 strains of Mycobacterium tuberculosis, 78 strains of non-acidic tuberculosis strain, and 68 strains of Mycobacteria were isolated from clinical specimens. The standard strain used was substantially the same as mentioned in Example 5-1.
임상검체에서 분리된 결핵균 186주, 항산성비결핵균 78주는 액체배지(MGIT 마이코박테리아 배지)나 고체(Ogawa배지)배지에서 검출되었거나 객담검체에서 직접 검출된 균주이었다. ATCC, KCTC 균주는 액체배지에서 배양하여 사용하였고, KMRC 균주는 고체배지에서 배양하여 사용하였다.186 strains of M. tuberculosis and 78 non-acidic tuberculosis strains isolated from clinical specimens were detected in liquid medium (MGIT mycobacterial medium) or solid (Ogawa medium) or directly in sputum specimens. ATCC and KCTC strains were used in culture in liquid medium, and KMRC strains were used in culture in solid medium.
액체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 균이 배양된 MGIT 마이코박테리아 배양튜브를 잘 섞은 후에 액체 배지 500㎕를 취해 1.5㎖ 튜브에 넣고 14,000 rpm로 5분간 원심분리 시켰다. 원심분리 후 상층액은 버리고 남은 침사부분에 멸균증류수 300㎕ 넣고 끓는 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm로 5분간 원심분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in liquid medium was extracted as follows. After mixing the bacteria cultured MGIT mycobacterial culture tube well, 500μl of liquid medium was taken into a 1.5ml tube and centrifuged at 14,000 rpm for 5 minutes. After centrifugation, the supernatant was discarded, and 300 µl of sterile distilled water was added to the remaining sedimentation portion and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
고체배지에서 자란 마이코박테리아의 DNA는 다음과 같이 추출하였다. 1.5㎖튜브에 멸균증류수 500㎕넣고 고체배지에서 1 백금이를 취해 멸균증류수에 풀었다. 이 튜브를 끓는 물에 10분간 중탕 가열한 후 14,000 rpm에서 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The DNA of mycobacteria grown in solid medium was extracted as follows. 500 µl of sterile distilled water was added to a 1.5 ml tube, and 1 platinum was taken from a solid medium and then dissolved in sterile distilled water. The tube was heated in boiling water for 10 minutes and then centrifuged at 14,000 rpm for 5 minutes to use the supernatant as template DNA for polymerase chain reaction.
객담검체는 다음과 같이 처리하였다. 15㎖ 혹은 50㎖ 튜브에 담긴 객담량과 동일한 량의 1N NaOH를 첨가하여 10분간 방치하여 객담을 액화시켰다. 14,000rpm에서 2분간 원심 분리하여 상층액을 버리고 남아 있는 침전물에 멸균증류수 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm으로 2분간 원심 분리하여 상층액을 제거하였다. 남아 있는 침전물에 멸균증류 1㎖를 넣고 10초간 잘 섞은 후 14,000rpm에서 2분간 원심 분리 한 후 상층액을 버렸다. 상층액을 제거하고 남아 있는 침전물에 5% chelex 수지(Biorad, USA) 100㎕와 10㎎/㎖ proteinase K 1㎕를 넣고 잘 섞어 주었다. 56℃에서 15분간 방치한 후 잘 혼합하여 끓은 물에 10분간 중탕 가열하였다. 중탕 가열 후 14,000 rpm 5분간 원심 분리 시켜서 상층액을 중합효소연쇄반응의 주형 DNA로 사용하였다.The sputum sample was processed as follows. Sputum was liquefied by adding 1N NaOH equal to the amount of sputum contained in a 15 ml or 50 ml tube and left for 10 minutes. The supernatant was discarded by centrifugation at 14,000 rpm for 2 minutes, 1 ml of sterile distilled water was added to the remaining precipitate, mixed well for 10 seconds, and centrifuged at 14,000 rpm for 2 minutes to remove the supernatant. 1 ml of sterile distillation was added to the remaining precipitate, mixed well for 10 seconds, centrifuged at 14,000 rpm for 2 minutes, and the supernatant was discarded. The supernatant was removed, and 100 μl of 5% chelex resin (Biorad, USA) and 1 μl of 10 mg / ml proteinase K were added to the remaining precipitates. After standing at 56 ° C. for 15 minutes, the mixture was mixed well and heated in boiling water for 10 minutes. Supernatant was used as template DNA for the polymerase chain reaction by centrifugation at 14,000 rpm for 5 minutes after heating the bath.
(2) 이중 실시간 중합효소연쇄반응법(2) Dual Real Time Polymerase Chain Reaction
Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA)를 사용하여 이중 실시간 중합효소연쇄반응을 실시하였다. 이중 실시간 중합효소연쇄반응은 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)을 이용하였다. 약 95℃에서 약 5분간 변성 공정을 1 사이클(cycle), 약 95℃에서 약 15초 간 변성, 약 65℃에서 약 15초 간 어닐링 및 연장 공정을 1 싸이클로 하여, 40 싸이클을 수행하였다. 이때, 이중 실시간 중합효소연쇄반응을 수행하는 반응물의 조성은 하기 표 7과 같다. 하기 primer-probes Mix에는 정방향 프라이머와 역방향 프라이머가 동일한 량(10pmole/㎕)으로 들어 있고 프루브는 4pmole/㎕이 있었다. 따라서 반응에 사용되는 MTC의 primer-probes mix 1.25㎕에는 정방향 및 역방향 프라이머가 각각 12.5pmole이 되고 프로브는 5pmole이 들어 있게 되었다. 총 25uL의 중합연쇄반응을 수행하는 반응물의 총부피가 25㎕이니 primer의 농도는 0.5uM(12.5pmoles/25㎕), 프로브는 0.2uM(5pmole/25㎕)가 사용되었다. NTM의 정방향 및 역방향 프라이머, 프로브의 농도는 MTC와 동일하게 사용되었다. Dual real-time polymerase chain reaction was performed using a Rotor-Gene multiplex PCR Kit (QIAGEN Inc., Germantown, MD, USA). Dual real time polymerase chain reaction was performed using Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). 40 cycles were performed using one cycle of the denaturation process at about 95 ° C. for about 5 minutes, one cycle of denaturation at about 95 ° C. for about 15 seconds, and annealing and extension processes at about 65 ° C. for about 15 seconds. At this time, the composition of the reaction to perform the double real-time polymerase chain reaction is shown in Table 7. In the following primer-probes Mix, the forward primer and the reverse primer contained the same amount (10 pmole / μl) and the probe was 4 pmole / μl. Therefore, 1.25 μl of the primer-probes mix of MTC used for the reaction had forward and reverse primers of 12.5 pmole and probes of 5 pmole. The total volume of 25uL of polymerase chain reaction was 25µL. The primer concentration was 0.5uM (12.5pmoles / 25µl) and the probe 0.2uM (5 pmole/25µl). The concentrations of the forward and reverse primers and probes of NTM were used the same as MTC.
표 7
성분 부피(㎕) 농도
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-Probes Mix 프라이머(10pmole/㎕) 1.25 0.5uM
프로브(4pmole/㎕) 0.2uM
Nuclease free water 6.25 -
샘플 DNA template 5 -
전체 25 -
TABLE 7
ingredient Volume (μl) density
2X Rotor-Gene Multiplex PCR Master Mix 12.5 1X
Primers-probes mix Primer (10 pmole / μl) 1.25 0.5 uM
Probe (4 pmole / μl) 0.2 uM
Nuclease free water 6.25 -
Sample DNA template 5 -
all 25 -
이중 실시간 중합효소연쇄반응 중 결합 및 연장단계에서 프로브에 의해 생성된 형광을 Rotor-Gene Q(QIAGEN Inc., Germantown, MD, USA)에서 측정한다. 상기 이중 실시간 중합효소연쇄반응 방법은 DNA 중합효소와 형광공명 에너지이동(Fluorescence Resonance Energy Transfer, FRET)의 원리에 의해 실시간 중합효소연쇄반응의 매 주기마다 실시간으로 시행되는 형광을 검출하고 정량하는 방법이다. 실시간 모니터 상에서 FAMTM 이 발색되는 경우 녹색채널(510±5nm), VICTM이 발색되는 경우 노랑채널(555±5nm)에서 표시하도록 지정하였다. 녹색채널(green channel)과 노랑채널(Yellow channel)에서 형광을 관찰하였다.Fluorescence generated by the probe in the binding and extension steps during the double real time polymerase chain reaction is measured in Rotor-Gene Q (QIAGEN Inc., Germantown, MD, USA). The dual real-time polymerase chain reaction method is a method for detecting and quantitating fluorescence performed in real time every cycle of the real-time polymerase chain reaction by the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). . On the real-time monitor, it was designated to display on the green channel (510 ± 5nm) when the FAM TM was developed and on the yellow channel (555 ± 5nm) when the VIC TM was developed. Fluorescence was observed in the green channel and the yellow channel.
3. 이중 실시간 중합효소연쇄반응법 수행 결과3. Result of Dual Real Time Polymerase Chain Reaction
도 37 내지 도 42는 상기 균주들의 이중 실시간 중합효소연쇄반응법에서 얻은 결과를 나타낸다. 상기 그래프들에서, x축은 중합효소연쇄반응의 싸이클 수이고 y축은 형광세기(fluoresence, F)이다. 도 37 및 도 38는 각각 결핵균(MTC)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 39 및 도 40는 각각 항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다. 도 41 및 도 42는 각각 결핵균(MTC)+항산성비결핵균(NTM)의 이중 실시간 중합효소연쇄반응의 녹색채널과 노랑채널에서의 중합효소연쇄반응의 싸이클 수에 대한 y축은 형광세기의 변화를 나타내는 그래프이다.37 to 42 show the results obtained in the double real time polymerase chain reaction method of the strains. In the graphs, the x-axis is the cycle number of the polymerase chain reaction and the y-axis is the fluorescence intensity (F). 37 and 38 are graphs showing changes in fluorescence intensity of cycle numbers of polymerase chain reaction in green and yellow channels of double real time polymerase chain reaction of Mycobacterium tuberculosis (MTC), respectively. 39 and 40 are graphs showing changes in fluorescence intensity of the cycle number of the polymerase chain reaction in the green channel and the yellow channel of the double-real-time polymerase chain reaction of NRT. 41 and 42 show y-axis for the number of cycles of polymerase chain reaction in the green channel and the yellow channel of the tubercle bacillus (MTC) + acidic non-tuberculosis bacterium (NTM), respectively. It is a graph.
도 37 내지 도 42를 참조하면, 결핵균(MTC)은 노랑채널에서 IS6110 유전자, 항산성비결핵균(NTM)은 녹색채널에서 16S rRNA 유전자, 결핵균(MTC)+항산성비결핵균(NTM)은 각각 노랑채널과 녹색채널에서 IS6110 유전자와 16S rRNA 유전자가 특이적으로 증폭되는 결과를 나타내어, 본 발명에 의한 프라이머 및 프로브를 이용할 경우, 이중 실시간 중합효소연쇄반응법으로 임상 검체에서 결핵균과 항산성비결핵균을 동시에 높은 신뢰성을 가지고 검출할 수 있음을 알 수 있었다.Referring to FIGS. 37 to 42, the MTC is an IS6110 gene in the yellow channel, the nonacidic tuberculosis bacterium (NTM) is the 16S rRNA gene in the green channel, the tuberculosis bacterium (MTC) + the acidic non-tuberculosis bacterium (NTM) is the yellow channel, respectively. The results show that the IS6110 gene and 16S rRNA gene are specifically amplified in the green channel. When using the primers and probes according to the present invention, the dual real-time polymerase chain reaction method results in high reliability of tuberculosis bacteria and anti-acidic tuberculosis bacteria in clinical specimens. It can be seen that can be detected with.
본 실시예들을 통하여, 결핵균 특이적 염기서열 및 결핵균에는 없는 항산성비결핵균의 고유염기서열을 정방향 프라이머 또는 역방향 프라이머; 및/또는 프로브로 디자인하여, 검사키트를 제작하여 이중 실시간 중합효소연쇄반응법으로, 상기 결핵균과 항산성비결핵균의 특이적 염기서열 탐색에 이용한 표준균주 들에 평가할 때, 높은 수준의 신뢰성으로 결핵균과 항산성비결핵균을 평가할 수 있는 수단임을 확인할 수 있었다. 따라서, 여러 종의 결핵균과 항상성비결핵균을 효과적으로 검출할 수 있는 수단을 제공할 수 있다.Through the present examples, the tuberculosis specific base sequence and the native nucleotide sequence of the anti-acidic non-tuberculosis tuberculosis which is absent from the tuberculosis bacterium may be forward or reverse primers; And / or designed with a probe, a test kit was prepared, and the double- real-time polymerase chain reaction method was performed to evaluate the standard strains used to search for specific sequences of the tuberculosis bacteria and the non-acidic tuberculosis bacteria. It was confirmed that it is a means for evaluating anti-acidic tuberculosis bacteria. Therefore, it is possible to provide a means for effectively detecting various kinds of Mycobacterium tuberculosis bacteria and homeostatic non-tuberculosis bacteria.
본 실시예들에 따르면, 결핵균과 항산성비결핵균에 특이적인 유전자 염기서열을 검출할 수 있는 결핵균과 항산성비결핵균 검출용 프라이머 및/또는 프로브는 결핵균과 항산성비결핵균에 대해 진단 감수성 및 특이성이 높다. 또한, 본 실시예들에 따른 결핵균과 항산성비결핵균 검출용 프라이머 및/또는 프로브를 이용하는 이중 실시간 중합효소연쇄반응법에 의하면 검체내에 결핵균과 항산성비결핵균을 동시에 보다 효율적으로 검출할 수 있는 임상진단 수단을 제공할 수 있다.According to the present embodiments, the primers and / or probes for detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria that can detect gene sequences specific for Mycobacterium tuberculosis and anti-acidic tuberculosis bacteria have high diagnostic susceptibility and specificity to Mycobacterium tuberculosis and anti-acidic tuberculosis bacteria. In addition, according to the dual real-time polymerase chain reaction method using the primers and / or probes for detecting the tuberculosis bacteria and anti-acidic non-tuberculosis bacterium according to the present embodiments, clinical diagnostic means capable of more efficiently detecting tuberculosis bacteria and anti-acidic tuberculosis bacteria in the sample at the same time Can be provided.
본 발명은 결핵균과 항산성비결핵균에 특이적인 유전자 염기서열을 검출할 수 있는 검출용 프라이머 세트 및/또는 프로브, 및 검출키트 및 검출 방법을 제공함으로, 결핵균 및/또는 항산성비결핵균을 동시에 보다 효율적으로 검출할 수 있는 임상진단 수단을 제공할 수 있고, 이를 병원, 연구소 등 다양한 산업 분야에 활용할 수 있을 것이다.The present invention provides a primer set and / or probe for detecting a gene sequence specific to Mycobacterium tuberculosis and Mycobacterium tuberculosis, and a detection kit and a method for detecting tuberculosis and / or mycobacterium tuberculosis. It is possible to provide a means of detecting clinical diagnostics, which may be used in various industries such as hospitals and research institutes.
염기서열 1은 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 1 is a forward primer specific for the IS6110 gene of Mycobacterium tuberculosis complex.
염기서열 2는 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 역방향 프라이머이다.SEQ ID NO: 2 is a reverse primer specific for the IS6110 gene of Mycobacterium tuberculosis complex.
염기서열 3은 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 프로브이다.SEQ ID NO: 3 is a probe specific for the IS6110 gene of Mycobacterium tuberculosis complex.
염기서열 4는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다. SEQ ID NO: 4 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 5는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머 (NTM-1)이다. SEQ ID NO: 5 is a reverse primer (NTM-1) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 6은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머 (NTM-1)이다. SEQ ID NO: 6 is a reverse primer (NTM-1) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 7은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머 (NTM-1)이다. SEQ ID NO: 7 is a reverse primer (NTM-1) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 8은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머 (NTM-2)이다. SEQ ID NO: 8 is a reverse primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 9는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다. SEQ ID NO: 9 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 10은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머 (NTM-2)이다. SEQ ID NO: 10 is a reverse primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 11은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머 (NTM-2)이다. SEQ ID NO: 11 is a reverse primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 12는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 12 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 13은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 13 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 14는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 14 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 15는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 15 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 16은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 16 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 17은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 17 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 18은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 18 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 19는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 19 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 20은 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 역방향 프라이머이다.SEQ ID NO: 20 is a reverse primer specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
염기서열 21은 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 프로브이다.SEQ ID NO: 21 is a probe specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
염기서열 22는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 22 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 23은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 23 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 24는 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 정방향 공통 프라이머 (forward universal primer)이다.SEQ ID NO: 24 is a forward universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 25는 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 역방향 공통 프라이머 (reverse universal primer)이다.SEQ ID NO: 25 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 26은 결핵균 (Mycobacterium tuberculosis complex)의 16S rRNA 유전자에 특이적인 프로브이다. SEQ ID NO: 26 is a probe specific for the 16S rRNA gene of Mycobacterium tuberculosis complex.
염기서열 27은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브 (NTM-1)이다. SEQ ID NO: 27 is a probe (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 28은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브 (NTM-2)이다. SEQ ID NO: 28 is a probe (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 29는 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 정방향 공통 프라이머 (forward universal primer)이다. SEQ ID NO: 29 is a forward universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 30은 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 정방향 공통 프라이머 (forward universal primer)이다. SEQ ID NO: 30 is a forward universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 31은 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 역방향 공통 프라이머 (reverse universal primer)이다.SEQ ID NO: 31 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 32는 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 역방향 공통 프라이머 (reverse universal primer)이다.SEQ ID NO: 32 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 33은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브 (NTM-2)이다. SEQ ID NO: 33 is a probe (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 34는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브 (NTM-2)이다. SEQ ID NO: 34 is a probe (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 35는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 35 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 36은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 역방향 프라이머이다.SEQ ID NO: 36 is a reverse primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 37은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 37 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 38은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 38 is a probe specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 39는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 39 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 40은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 40 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 41은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 41 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 42는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 42 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 43은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 43 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 44는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 44 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 45는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 45 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 46은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 46 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 47은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 47 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 48은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 48 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 49는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 49 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 50은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 50 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 51은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 51 is a forward primer specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 52는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 52 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 53은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 53 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 54는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 54 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 55는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 55 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 56은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 56 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 57은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 57 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 58은 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 58 is a forward primer specific for the IS6110 gene of Mycobacterium tuberculosis complex.
염기서열 59는 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 역방향 프라이머이다.SEQ ID NO: 59 is a reverse primer specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
염기서열 60은 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 프로브이다.SEQ ID NO: 60 is a probe specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
염기서열 61은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머이다.SEQ ID NO: 61 is a forward primer specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 62는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 62 is a probe specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 63은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 63 is a probe specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 64는 결핵균 (Mycobacterium tuberculosis complex)의 IS6110 유전자에 특이적인 프로브이다.SEQ ID NO: 64 is a probe specific for the IS6110 gene of the Mycobacterium tuberculosis complex.
염기서열 65는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머(NTM-1)이다.SEQ ID NO: 65 is a forward primer (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 66은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머(NTM-2)이다.SEQ ID NO: 66 is a forward primer (NTM-2) specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 67은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머(NTM-3)이다.SEQ ID NO: 67 is a forward primer (NTM-3) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 68은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 68 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 69는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머 (NTM-1)이다.SEQ ID NO: 69 is a forward primer (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 70은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머 (NTM-1)이다.SEQ ID NO: 70 is a forward primer (NTM-1) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 71은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머 (NTM-2)이다.SEQ ID NO: 71 is a forward primer (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 72는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 정방향 프라이머 (NTM-2)이다.SEQ ID NO: 72 is a forward primer (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 73은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 73 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 74는 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브이다.SEQ ID NO: 74 is a probe specific for the 16S rRNA gene of Nontuberculous mycobacteria.
염기서열 75는 마이코박테이엄 (Mycobacterium)의 16S rRNA 유전자에 특이적인 역방향 공통 프라이머 (reverse universal primer)이다.SEQ ID NO: 75 is a reverse universal primer specific for 16S rRNA gene of Mycobacterium.
염기서열 76은 항산성비결핵균 (Nontuberculous mycobacteria)의 16S rRNA 유전자에 특이적인 프로브(NTM-2)이다.SEQ ID NO: 76 is a probe (NTM-2) specific for 16S rRNA gene of Nontuberculous mycobacteria.

Claims (34)

  1. 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.Probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 9.
  2. 제 1항에 있어서, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ(molecular grove binding non-fluorescence quencher)로 이루어진 군으로부터 선택된 1종의 형광 억제 물질(Quencher)로 표지된 것을 특징으로 하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.The method according to claim 1, wherein the 5 'end of the probe is one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled, and the 3 'end is labeled with one fluorescence inhibitor (Quencher) selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and molecular grove binding non-fluorescence quencher (MGGBFQ). Probe for detecting acidic non-tuberculosis 16S rRNA gene.
  3. 염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트;A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
    염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브;A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3;
    염기서열 4의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And
    염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising a probe for detecting the acidic non-tuberculosis 16S rRNA gene of SEQ ID NO: 9.
  4. 제 3항에 있어서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,The 5 'terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is one selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of 3 ′, and labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, characterized in that the 5 'end of the probe for detecting the IS6110 gene of the tuberculosis and 5' end of the non-acidic tuberculosis 16S rRNA gene detection probe is labeled with different fluorescent labeling factors.
  5. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 1의 정방향 프라이머 및 염기서열 2의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 3의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 4의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 9의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 3; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 4, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-tuberculosis bacterium 16S rRNA gene of SEQ ID NO: 9; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
  6. 염기서열 22의 정방향 프라이머;A forward primer of nucleotide sequence 22;
    염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머; 및One or two or more reverse primers selected from the group consisting of a primer of SEQ ID NO: 5, a primer of SEQ ID NO: 6, and a primer of SEQ ID NO: 7; And
    염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트.A primer set specific for 16S rRNA gene of non-acidic mycobacterium tuberculosis comprising a reverse primer of nucleotide sequence 8.
  7. 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.A probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 23.
  8. 제 7항에 있어서, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.8. The fluorescent labeling agent of claim 7, wherein the 5 ′ end of the probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Probe for detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene, characterized in that the 3 'end is labeled with one fluorescent inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3.
  9. 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트;A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20;
    염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브;A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21;
    염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primer sets specific for rRNA genes; And
    염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising a probe for detecting the acidic non-tuberculosis 16S rRNA gene of SEQ ID NO: 23.
  10. 제 9항에 있어서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질(Quencher)로 표지되고,10. The method according to claim 9, wherein the 5 ′ terminal of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of 3 ′, and labeled with one fluorescent inhibitor (Quencher) selected from the group consisting of 6-TAMRA and BHQ-1,2,3,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3,
    상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, characterized in that the 5 'end of the probe for detecting the IS6110 gene of the tuberculosis and 5' end of the non-acidic tuberculosis 16S rRNA gene detection probe is labeled with different fluorescent labeling factors.
  11. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 22의 정방향 프라이머, 염기서열 5의 프라이머, 염기서열 6의 프라이머 및 염기서열 7의 프라이머로 이루어진 군에서 선택된 하나 또는 둘 이상의 역방향 프라이머 및 염기서열 8의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머; 및 염기서열 23의 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; 16S of the anti-acidic tuberculosis bacterium comprising one or more reverse primers selected from the group consisting of a forward primer of nucleotide sequence 22, a primer of nucleotide sequence 5, a primer of nucleotide sequence 6 and a primer of nucleotide sequence 7 and a reverse primer of nucleotide sequence 8 primers specific for rRNA genes; And performing double real-time polymerase chain reaction of the DNA using a probe for detecting an acidic non-TB bacterium 16S rRNA gene of SEQ ID NO: 23; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
  12. 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브.Probe for detection of Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26.
  13. 제 12항에 있어서, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 하는 결핵균 16S rRNA 유전자 탐지용 프로브.The method of claim 12, wherein the 5 ′ end of the probe is one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. A probe for detecting Mycobacterium tuberculosis 16S rRNA gene, characterized in that the 3 'end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  14. 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.Probe for the detection of non-acidic Mycobacterium tuberculosis 16S rRNA gene comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28.
  15. 제 14항에 있어서, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.15. The fluorescence labeling factor of claim 14, wherein the 5 'end of the probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Probe for detecting the anti-acidic mycobacterium tuberculosis 16S rRNA gene, characterized in that the 3 'end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  16. 염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트;A common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25;
    염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브; 및Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And
    염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28.
  17. 제 16항에 있어서, 상기 결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,The method of claim 16, wherein the 5 'end of the Mycobacterium tuberculosis 16S rRNA gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of the species, the 3 ′ end is labeled with one fluorescent inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 결핵균의 16S rRNA 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, characterized in that the 5 'end of the 16S rRNA gene detection probe of the Mycobacterium tuberculosis and 5' end of the 16S rRNA gene detection probe labeled with different fluorescent labeling factors.
  18. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 24의 정방향 프라이머 및 염기서열 25의 역방향 프라이머를 포함하는 결핵균 및 항산성비결핵균의 16S rRNA 유전자를 증폭하기 위한 공통 프라이머 세트; 염기서열 26의 결핵균 16S rRNA 유전자 탐지용 프로브; 및 염기서열 27의 프로브 및 염기서열 28의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A common primer set for amplifying 16S rRNA genes of Mycobacterium tuberculosis and nonacidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 25; Probe for detecting Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 26; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 28; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
  19. 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.A probe for detecting non-acidic Mycobacterium tuberculosis 16S rRNA gene selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39.
  20. 제 19항에 있어서, 상기 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지된 것을 특징으로 하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브.The method of claim 19, wherein the 5 ′ end of the probe is one fluorescent labeling factor selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. A probe for detecting an acid-free M. tuberculosis 16S rRNA gene, characterized in that the 3 'end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ.
  21. 염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트;A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20;
    염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브;A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21;
    염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And
    염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the group consisting of a probe of nucleotide sequence 37, a probe of nucleotide sequence 38 and a nucleotide sequence 39 probe.
  22. 제 21항에 있어서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질(Quencher)로 표지되고,22. The method according to claim 21, wherein the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of 3 ′, and labeled with one fluorescent inhibitor (Quencher) selected from the group consisting of 6-TAMRA and BHQ-1,2,3,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA 및 BHQ-1,2,3으로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA and BHQ-1,2,3,
    상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, characterized in that the 5 'end of the probe for detecting the IS6110 gene of the tuberculosis and 5' end of the non-acidic tuberculosis 16S rRNA gene detection probe is labeled with different fluorescent labeling factors.
  23. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 1의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 35의 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 37의 프로브, 염기서열 38의 프로브 및 염기서열 39의 프로브로 이루어진 군으로부터 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 21; A primer set specific for the 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from the group consisting of a probe of SEQ ID NO: 37, a probe of SEQ ID NO: 38, and a probe of SEQ ID NO: 39; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
  24. 염기서열 61의 정방향 프라이머; 및Forward primer of SEQ ID NO: 61; And
    염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트.A primer set specific for 16S rRNA gene of non-acidic Mycobacterium tuberculosis comprising a reverse primer comprising a primer of SEQ ID NO: 5 and a primer of SEQ ID NO: 8.
  25. 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트;A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59;
    염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브;A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60;
    염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And
    염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a nucleotide sequence 62 probe or a nucleotide sequence 63 probe.
  26. 제 25항에 있어서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,The 1 type according to claim 25, wherein the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of 3 ′, and labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.The 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe and the 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe are labeled with different fluorescent labeling factors.
  27. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 60의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 61의 정방향 프라이머 및 염기서열 5의 프라이머 및 염기서열 8의 프라이머를 포함하는 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 62의 프로브 또는 염기서열 63의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 60; A primer set specific for 16S rRNA gene of non-acidic tuberculosis bacterium comprising a forward primer of nucleotide sequence 61 and a reverse primer comprising a primer of nucleotide sequence 5 and a primer of nucleotide sequence 8; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 62 or a probe of SEQ ID NO: 63; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
  28. 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머; 및A forward primer comprising a primer of nucleotide sequence 65, a primer of nucleotide sequence 66 and a primer of nucleotide sequence 67; And
    염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트.A primer set specific for 16S rRNA gene of non-acidic mycobacterium tuberculosis comprising a reverse primer of nucleotide sequence 36.
  29. 염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트;A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20;
    염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브;A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64;
    염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And
    염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis 16S rRNA gene detection probe selected from the probe of SEQ ID NO: 39 or the probe of SEQ ID NO: 68.
  30. 제 29항에 있어서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,30. The method according to claim 29, wherein the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of 3 ′, and labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.The 5 'end of the Mycobacterium tuberculosis IS6110 gene detection probe and the 5' end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe are labeled with different fluorescent labeling factors.
  31. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 58의 정방향 프라이머 및 염기서열 20의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 21의 프로브 또는 염기서열 64의 프로브에서 선택되는 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 65의 프라이머, 염기서열 66의 프라이머 및 염기서열 67의 프라이머를 포함하는 정방향 프라이머 및 염기서열 36의 역방향 프라이머를 포함하는 항산성비결핵균의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 39의 프로브 또는 염기서열 68의 프로브에서 선택되는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 20; A probe for detecting the Mycobacterium tuberculosis IS6110 gene selected from a probe of SEQ ID NO: 21 or a probe of SEQ ID NO: 64; A primer set specific for 16S rRNA gene of anti-acidic tuberculosis bacterium comprising a primer of SEQ ID NO: 65, a primer of SEQ ID NO: 66, and a primer of SEQ ID NO: 67; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe selected from a probe of SEQ ID NO: 39 or a probe of SEQ ID NO: 68; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
  32. 염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트;A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59;
    염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브;Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60;
    염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트; 및A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And
    염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 포함하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit comprising an anti-acidic tuberculosis bacterium 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76.
  33. 제 32항에 있어서, 상기 결핵균 IS6110 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,33. The method according to claim 32, wherein the 5 ′ end of the Mycobacterium tuberculosis IS6110 gene detection probe is selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 and NED. Labeled with a fluorescent labeling factor of 3 ′, and labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670 및 NED로 이루어진 군으로부터 선택되는 1종의 형광 표지 인자로 표지되며, 3′ 말단이 6-TAMRA, BHQ-1,2,3 및 MGBNFQ로 이루어진 군으로부터 선택된 1종의 형광 억제 물질로 표지되고,One fluorescent label selected from the group consisting of FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXAS RED, RED670, and NED at the 5 ′ end of the anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe. Labeled with a factor, and the 3 ′ end is labeled with one fluorescence inhibitor selected from the group consisting of 6-TAMRA, BHQ-1,2,3 and MGBNFQ,
    상기 결핵균의 IS6110 유전자 탐지용 프로브의 5′ 말단과 상기 항산성비결핵균 16S rRNA 유전자 탐지용 프로브의 5′ 말단이 서로 다른 형광 표지 인자로 표지된 것을 특징으로 하는 결핵균과 항산성비결핵균 검출 키트.Mycobacterium tuberculosis and anti-acidic tuberculosis detection kit, characterized in that the 5 'end of the probe for detecting the IS6110 gene of the tuberculosis and 5' end of the non-acidic tuberculosis 16S rRNA gene detection probe is labeled with different fluorescent labeling factors.
  34. 검체시료로부터 DNA를 분리하는 단계;Separating DNA from the sample;
    염기서열 58의 정방향 프라이머 및 염기서열 59의 역방향 프라이머를 포함하는 결핵균의 IS6110 유전자에 특이적인 프라이머 세트; 염기서열 60의 결핵균 IS6110 유전자 탐지용 프로브; 염기서열 24의 정방향 프라이머 및 염기서열 75의 역방향 프라이머를 포함하는 마이코박테리아의 16S rRNA 유전자에 특이적인 프라이머 세트; 및 염기서열 27의 프로브 및 염기서열 76의 프로브를 포함하는 항산성비결핵균 16S rRNA 유전자 탐지용 프로브를 사용하여 상기 DNA를 이중 실시간 중합효소연쇄반응을 시키는 단계; 및A primer set specific for the IS6110 gene of Mycobacterium tuberculosis comprising a forward primer of SEQ ID NO: 58 and a reverse primer of SEQ ID NO: 59; Probe for detecting Mycobacterium tuberculosis IS6110 gene of SEQ ID NO: 60; A primer set specific for the mycobacterial 16S rRNA gene comprising a forward primer of SEQ ID NO: 24 and a reverse primer of SEQ ID NO: 75; And performing double real-time polymerase chain reaction of the DNA using an anti-acidic Mycobacterium tuberculosis 16S rRNA gene detection probe comprising a probe of SEQ ID NO: 27 and a probe of SEQ ID NO: 76; And
    상기 이중 실시간 중합효소연쇄반응 결과를 확인하는 단계를 포함하는 결핵균과 항산성비결핵균을 검출하는 방법.Method for detecting tuberculosis bacteria and non-acidic tuberculosis bacteria comprising the step of confirming the dual real-time polymerase chain reaction results.
PCT/KR2011/003903 2010-05-27 2011-05-27 Method for detecting mycobacterium tubericulosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction WO2011149305A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US13/700,157 US20130210005A1 (en) 2010-05-27 2011-05-27 Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction
CN201180036998.5A CN103038348B (en) 2010-05-27 2011-05-27 Method for detecting mycobacterium tubericulosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
KR10-2010-0049692 2010-05-27
KR20100049692 2010-05-27
KR20100062847 2010-06-30
KR10-2010-0062847 2010-06-30
KR1020110009530A KR101158649B1 (en) 2010-05-27 2011-01-31 Methods for identification and detection of mycobacterium tuberculosis complex and nontuberculous mycobacteria using duplex real time polymerase chain reaction
KR10-2011-0009530 2011-01-31

Publications (3)

Publication Number Publication Date
WO2011149305A2 WO2011149305A2 (en) 2011-12-01
WO2011149305A9 true WO2011149305A9 (en) 2012-03-01
WO2011149305A3 WO2011149305A3 (en) 2012-05-18

Family

ID=45004597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2011/003903 WO2011149305A2 (en) 2010-05-27 2011-05-27 Method for detecting mycobacterium tubericulosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction

Country Status (1)

Country Link
WO (1) WO2011149305A2 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101403507B1 (en) * 2013-03-21 2014-06-09 주식회사 현일바이오 Methods for Selectively Detecting Mycobacterium tuberculosis complex and Nontuberculous mycobacteria and Kits Using the Same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100234975B1 (en) * 1997-07-28 1999-12-15 국윤호 Identification of mycobacterial species by pcr-rflp of the rna polymerase gene(rpob)
KR100968069B1 (en) * 2007-10-24 2010-07-08 대한민국 Protein antigen for diagnosis of Bovine tuberculosis and its purification method

Also Published As

Publication number Publication date
WO2011149305A3 (en) 2012-05-18
WO2011149305A2 (en) 2011-12-01

Similar Documents

Publication Publication Date Title
WO2018001258A1 (en) Probe for nucleic acid enrichment and capture, and design method thereof
WO2011157222A1 (en) Joint detection methods for lymphoma fusion genes and diagnostic kits thereof
WO2019124904A1 (en) Microfluidic paper chip for detecting micro-organism, method for preparing the same and method for detecting micro-organism using the same
WO2018139826A1 (en) Age predicting method using dna methylation
WO2020105873A1 (en) Dignostic kit and whole genome sequence identification method which use amplification of whole genome of human alpha coronavirus
WO2012048607A1 (en) Method for detecting pca3 and psa genes, and diagnostic kit thereof
WO2018038352A2 (en) Autoantibody biomarker for diagnosing dementia and method for diagnosing dementia by using same
WO2021182881A1 (en) Multiple biomarkers for breast cancer diagnosis and use thereof
WO2011149305A9 (en) Method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction
WO2011120398A1 (en) Joint detection method for leukemia fusion genes and diagnostic kits thereof
WO2021230663A1 (en) Method for predicting prognosis of patients having early breast cancer
WO2019135477A2 (en) Method of diagnosing tsutsugamushi disease by using multicopy gene
WO2015167087A1 (en) Method for predicting risk of ankylosing spondylitis using dna copy number variants
WO2014157825A1 (en) Method and kit for testing hla alleles using speed multiplex pcr
WO2023027418A1 (en) Composition for detecting oral disease-causing bacteria, or use thereof
WO2011139032A2 (en) Primer composition for amplifying a gene region having diverse variations in a target gene
WO2023191206A1 (en) Exploratory data analysis automation system and method based on variable attributes
WO2012050353A2 (en) Method whereby the base sequence of a target polynucleotide is non-specifically extended
WO2010140827A9 (en) Automatic detection kit for hla alleles by real-time polymerase chain reaction
WO2019103475A2 (en) Hla genotyping kit using real-time polymerase chain reaction
WO2014175604A2 (en) Composition for diagnosing avellino corneal dystrophy and method for diagnosing avellino corneal dystrophy
WO2020122679A1 (en) Method for detecting a target analyte in a sample using an s-shaped function for a slope data set
WO2019022520A1 (en) Nucleic acid complex pair, competitive structure, and pcr kit using both
EP4204586A1 (en) Method for positive control reaction using pre-positive control composition
WO2009154357A2 (en) Human papilloma virus detection method and detection kit

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201180036998.5

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11786937

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 13700157

Country of ref document: US

122 Ep: pct application non-entry in european phase

Ref document number: 11786937

Country of ref document: EP

Kind code of ref document: A2