WO2011149305A2 - Procédé de détection de mycobacterium tubericulosis et de mycobactéries non tuberculeuses au moyen d'une double réaction en chaîne de la polymérase en temps réel - Google Patents

Procédé de détection de mycobacterium tubericulosis et de mycobactéries non tuberculeuses au moyen d'une double réaction en chaîne de la polymérase en temps réel Download PDF

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WO2011149305A2
WO2011149305A2 PCT/KR2011/003903 KR2011003903W WO2011149305A2 WO 2011149305 A2 WO2011149305 A2 WO 2011149305A2 KR 2011003903 W KR2011003903 W KR 2011003903W WO 2011149305 A2 WO2011149305 A2 WO 2011149305A2
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seq
probe
primer
tuberculosis
mycobacterium tuberculosis
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PCT/KR2011/003903
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English (en)
Korean (ko)
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WO2011149305A9 (fr
WO2011149305A3 (fr
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김정욱
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울산대학교 산학협력단
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Priority claimed from KR1020110009530A external-priority patent/KR101158649B1/ko
Application filed by 울산대학교 산학협력단 filed Critical 울산대학교 산학협력단
Priority to CN201180036998.5A priority Critical patent/CN103038348B/zh
Priority to US13/700,157 priority patent/US20130210005A1/en
Publication of WO2011149305A2 publication Critical patent/WO2011149305A2/fr
Publication of WO2011149305A9 publication Critical patent/WO2011149305A9/fr
Publication of WO2011149305A3 publication Critical patent/WO2011149305A3/fr

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    • 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 the detection of Mycobacterium tuberculosis bacteria and nonacidic 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. Perception 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 method of rapid and accurate detection of tuberculosis bacillus and acidic non-tuberculosis bacillus for selecting therapeutic drugs 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 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 an acidic non-TB 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 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 types of Mycobacterium tuberculosis complex (MTC).
  • 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 to 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.
  • a probe for detecting the non-acidic Mycobacterium tuberculosis 16S rRNA gene of SEQ ID NO: 23 is provided.
  • 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 ′ end of the probe is one kind of 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 the detection of 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 antiacidic non-tuberculosis bacterium comprising a forward primer of SEQ ID NO: 24 and a reverse primer set of SEQ ID NO: 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 has 5′-FAM-tggaaagcgtttggtagc-MGB-3 ′ (base sequence 33) and 5′-FAM-tggaaagtgtttggtagc-MGB-3 ′ (base sequence 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 includes an anti primer 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 markers, light emitting materials, bioluminescent materials, isotopes, and the like, which are commonly used, but are not limited thereto. If the excitation and emission wavelengths are different depending on the type of fluorescent labeling factors, the method of use is also different. , 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
  • 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 types of Mycobacterium tuberculosis complex (MTC).
  • 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 to the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the various types of Mycobacterium tuberculosis complex (MTC).
  • 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 the 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 to the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the various types of Mycobacterium tuberculosis complex (MTC).
  • 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 to the IS6110 gene of the Mycobacterium tuberculosis bacterium was designed to detect all the various types of Mycobacterium tuberculosis complex (MTC).
  • 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 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).
  • NCBI National Center for Biotechnology Information
  • 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 from clinical specimens.
  • 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 from clinical specimens.
  • 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 sap. 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 from clinical specimens.
  • 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 from clinical specimens.
  • 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 sap. 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 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.
  • 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 Bacteria
  • 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 from clinical specimens.
  • 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 from clinical specimens.
  • 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 sap. 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 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. Accordingly, 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 the nucleotide sequence 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 sap. 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 the 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 sap. 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 sap. 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 reactants performing 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 the probe for detecting Mycobacterium tuberculosis (MTC), and the Taqman probe of SEQ ID NO: 68 was used as the probe for the detection of non-acidic tuberculosis bacteria. Polymerase chain reaction was performed.
  • MTC Mycobacterium tuberculosis
  • SEQ ID NO: 68 was used as the probe for the detection of non-acidic 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 sequence 68 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: 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 ′ (base sequence 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 sap. 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 reactant 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 a forward primer or a reverse primer; 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 non-tuberculosis bacteria have high diagnostic susceptibility and specificity to Mycobacterium tuberculosis and acidic non-TB 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 for detecting clinical diagnosis, which can 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.

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Abstract

La présente invention concerne : un ensemble d'amorces et/ou une sonde pour la détection de Mycobacterium tubericulosis et de mycobactéries non tuberculeuses pour un gène IS6110 et/ou gène d'ARNr 16S spécifiques de Mycobacterium tubericulosis et pour un gène d'ARNr 16S spécifique de mycobactéries non tuberculeuses; une trousse de détection de Mycobacterium tubericulosis et de mycobactéries non tuberculeuses, contenant les mêmes; et un procédé de détection de Mycobacterium tubericulosis et de mycobactéries non tuberculeuses au moyen d'une double réaction en chaîne de la polymérase en temps réel utilisant les mêmes. La présente invention offre des moyens de diagnostic clinique permettant de détecter et d'analyser efficacement et simultanément Mycobacterium tubericulosis et des mycobactéries non tuberculeuses.
PCT/KR2011/003903 2010-05-27 2011-05-27 Procédé de détection de mycobacterium tubericulosis et de mycobactéries non tuberculeuses au moyen d'une double réaction en chaîne de la polymérase en temps réel WO2011149305A2 (fr)

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CN201180036998.5A CN103038348B (zh) 2010-05-27 2011-05-27 通过使用双重实时聚合酶链式反应对结核分枝杆菌和非结核分枝杆菌进行检测的方法
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

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KR20100049692 2010-05-27
KR10-2010-0049692 2010-05-27
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KR1020110009530A KR101158649B1 (ko) 2010-05-27 2011-01-31 이중 실시간 중합효소연쇄반응법을 이용한 결핵균과 항산성비결핵균의 검출 방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2977465A4 (fr) * 2013-03-21 2016-11-16 Hyunil Bio Co Procédé de détection sélective pour mycobacterium tuberculosis et des mycobactéries non tuberculeuses et kit utilisant celui-ci

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR970065731A (ko) * 1997-07-28 1997-10-13 국윤호 마이코박테리아의 rpoB 유전자 분절을 표적으로 하는 PCR-RFLP에 의한 마이코박테리아 균종의 탐지 및 동정방법
KR20090041663A (ko) * 2007-10-24 2009-04-29 대한민국(관리부서 : 농림수산식품부 국립수의과학검역원) 우결핵균 특이단백질 및 그 정제방법

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR970065731A (ko) * 1997-07-28 1997-10-13 국윤호 마이코박테리아의 rpoB 유전자 분절을 표적으로 하는 PCR-RFLP에 의한 마이코박테리아 균종의 탐지 및 동정방법
KR20090041663A (ko) * 2007-10-24 2009-04-29 대한민국(관리부서 : 농림수산식품부 국립수의과학검역원) 우결핵균 특이단백질 및 그 정제방법

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE NCBI, GENBANK 02 December 2009 'Mycobacterium bovis BCG strain M140 16S ribosomal RNA gene, partial sequence' Database accession no. GU142938 *
DATABASE NCBI, GENBANK 10 November 2009 'Mycobacterium sp. GN-01 16S ribosomal RNA gene, partial sequence' Database accession no. GU084182 *
GILBERT, Y. ET AL.: 'Metalworking fluids biodiversity characterization' JOURNAL OF APPLIED MICROBIOLOGY vol. 108, 15 June 2009, pages 437 - 449 *
NAIR, N. ET AL.: 'Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants' MOLECULAR MICROBIOLOGY vol. 71, no. 2, 19 November 2008, pages 291 - 304 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2977465A4 (fr) * 2013-03-21 2016-11-16 Hyunil Bio Co Procédé de détection sélective pour mycobacterium tuberculosis et des mycobactéries non tuberculeuses et kit utilisant celui-ci
US10301686B2 (en) 2013-03-21 2019-05-28 Hyunil-Bio Co. Selective detection method for Mycobacterium tuberculosis and nontuberculous mycobacteria and kit using same

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