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  • 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
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • 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
• • 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
  • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

• the present invention relates to a composition for detection of Mycobacterium tuberculosis complex or Mycobacterium genus and a method for simultaneous detection of M tuberculosis complex and Mycobacterium genus by multiplex real-time PCR using the same. More specifically, the present invention relates to a composition for detection of M, tuberculosis complex or Mycobacterium genus, comprising (i) a primer and/or probe targeting IS6110 that is an M tuberculosis complex-specific gene, ( ⁇ ) a primer and/or probe targeting rpoB that is a Mycobacterium genus-specific gene, and optionally (in) a primer and/or probe targeting a plant- derived gene as an internal control.
• Tuberculosis is the world's leading infectious cause of death since before recorded history.
• the causative bacterial pathogen of tuberculosis is tuberculosis-causing bacteria which belong to the Mycobacterium genus and have a rod (bacillus) morphology, a thickness of 0.2-0.5 m and a length of 1-4 ⁇ sa.
• the Mycobacterium genus encompasses a variety of pathogenic species causing respiratory diseases (including tuberculosis), leprosy (Hansen's disease), etc. in humans and animals. Up to date, more than 100 mycobacterial species have been identified (Shinnick TM et al, Mycobacterial Taxonomy. Eur J Clin Microbiol Infect Dis. 1994;13(11):884-901).
• Mycobacterium tuberculosis is known as a major bacterial strain causing tuberculosis in humans. Rare cases of human tuberculosis are caused by Mycobacterium bovis. Mycobacterium leprae is known to cause leprosy, i.e. Hansen's disease (Shinnick TM and Good RC. Mycobacterial Taxonomy. Eur J Clin Microbiol Infect Dis. 1994;13(11):884-901).
• Tuberculosis is one of the major causes of illness and death in economically underdeveloped countries. In Korea, it is estimated that 120,000 people are annually infected with tuberculosis pathogens. According to the statistical data of the Korea National Statistical Office (KNSO) in 2004, the incidence of new tuberculosis patients amounted up to 30,687 cases (64 cases per 100,000 people) in the survey of 2003, and Korea was shamefully ranked the highest among the 30 OECD Member countries in terms of tuberculosis mortality.
• KNSO National Statistical Office
• nontuberculous mycobacteria may include M. avium-intracellulare complex or M. avium complex, M. fortuitum, M. chelonae, M. gordonae, M. szulagai, M kansasii, M ⁇ icanum, and M genavense (Barnes PF et al., Tuberculosis in patients with human immunodeficiency virus infectioa N Engl J Med. 1991;324(23): 1644-1650).
• M. avium complex which is the most common species of NTM, is known to exhibit 10 to 100-fold lower susceptibility to first- line anti-tuberculosis drugs, when compared with tuberculosis bacteria For this reason, the American Thoracic Society (ATS) presents a guideline for diagnosis and treatment of NTM diseases (American Thoracic Society, Diagnosis and treatment of disease caused by nontuberculous mycobacteria Am J Respir Crit Care Med. 1997; 156(2): S 1-S25).
• ATS American Thoracic Society
• NTM diseases are very similar to those of tuberculosis infections, but therapeutic drugs for treatment of both diseases may be completely different therebetween.
• therapeutic drugs for treatment of both diseases may be completely different therebetween.
• tuberculosis may include examination of patients' clinical symptoms, tuberculin skin test, X-ray photography and bacteriological test.
• the tuberculin skin test is the simplest means for identifying patients infected with tuberculosis, but disadvantageously may often produce false-negative results under anergy-inducing conditions due to severe tuberculosis, rubeola (measles) and immunosuppression.
• the efficacy of X-ray diagnosis is determined by the reader's ability and an average of 25% of patients diagnosed positive by X-ray examination are diagnosed negative by other diagnostic tests.
• the diagnosis of tuberculosis by X-ray photography depends on the finding of abnormal shadows and accurate interpretation of the abnormal shadows by the reader.
• the bacteriological test Ie. the detection of tuberculosis bacteria, is a reliable method for diagnosis of tuberculosis infection and may include a smear test, a culture test, a molecular diagnostic test, etc.
• the smear test generally employs Ziehl-Neelsen staining which is a special technique used to identify acid-fast organisms, mainly mycobacteria. Although it is a simple and rapid diagnostic technique, identification of tuberculosis and nontuberculous mycobacteria is difficult and detection sensitivity is also low.
• the culture test is advantageous to provide high detection sensitivity for accurate diagnosis and identification of tuberculosis bacteria That is, it is possible to detect pathogenic bacteria even at a very low concentration of about 10 bacteria/mL of a test sample.
• this method is not therapeutically favorable for the treatment of tuberculosis patients, due to requirements for long-term culture of about 4 to 8 weeks and examination by well-trained specialists to get the test results.
• a BACTEC method (Becton Dickinson, USA) is a novel method for diagnosis of tuberculosis that radioisotopicaUy measures an amount of 14 CO 2 produced by the metabolism of bacteria inoculated onto a liquid medium containing C 14 -palmitate and expresses the measured amount as a growth quotient. This method takes an average of 16 days to get the results. However, there is a problem that facilities and skilled persons for safe handling and management of radioisotopes are required.
• PCR polymerase chain reaction
• PCR assay has shortcomings such as high risk of carry-over contamination and need for PCR technicians (Noordhoek GT et al., Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol. 1994;32(2):277-284).
• Ihe PCR assay exhibits high sensitivity and specificity particularly in the smear-positive sample. Therefore, the possibility should be considered that the sample of interest is likely to contain NTM when it is acid-fast smear-positive and PCR- negative.
• NTM infection takes a culture period of 4 to 8 weeks (Centers for Disease Control and Prevention (CDC). Update: Nucleic acid amplification tests for tuberculosis. MMWR Morb Mortal WkIy Rep
• PCR-RFLP polymerase chain reaction-restriction fragment length polymorphism
• the present invention has been made in view of the above problems, and it is an object of the present invention to provide a primer and/or probe having excellent sensitivity for an M. tuberculosis complex-specific gene IS6110, which is intended for accurate diagnosis of M tuberculosis.
• NTM nontuberculous mycobacteria
• FIG. 1 is a table depicting partial base sequences of rpoB genes of mycobacterial species conventionally known in the art
• FIG. 2 is a photograph showing the multiplex real-time PCR results of M tuberculosis complex-positive samples using an analysis composition in accordance with the present invention
• FIG. 3 is a photograph showing the multiplex real-time PCR results of Mycobacterium genus-positive samples using an analysis composition in accordance with the present invention.
• FIG. 4 is a photograph showing the multiplex real-time PCR results of negative samples using an analysis composition in accordance with the present invention.
• the present invention provides a composition for detection of M. tuberculosis complex or non-tuberculosis mycobacteria, comprising at least one primer selected from the group consisting of: a primer containing a base sequence as set forth in SEQ ID NO: 1 ; a primer containing a base sequence as set forth in SEQ ID NO: 2; a primer containing a base sequence as set forth in SEQ ID NO: 3 ; and a primer containing a base sequence as set forth in SEQ ID NO: 4.
• a primer containing a base sequence as set forth in SEQ ID NO: x is intended to encompass a base sequence with a sequence homology of 95% or more. The reason is as follows. If two or more bases are altered when a primer is 20bp in length, lowering of a melting temperature of the primer is 5 ° C or more, which consequently leads to undesirable results. On the other hand, if a single base is changed, it is possible to obtain substantially the same results by slightly lowering an annealing temperature in a PCR process.
• a primer containing the base sequence of SEQ ID NO: x is simply designated as "a primer of SEQ ID NO: x.
• the primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 are primers that are intended to target an IS6110 gene region which is an M. tuberculosis complex-specific gene region.
• tuberculosis bacteria means human-type tuberculosis bacilli, Ie. Mycobacterium tuberculosis in a narrow sense
• this term in the context of the present invention is intended to encompass Mycobacterium bovis, Mycobacterium microti and Mycobacterium qfricanum as well as M. tuberculosis in a broad sense and is used interchangeably with the term “M tuberculosis complex” or "TB complex", if necessary.
• the IS6110 gene is an insertion sequence which is found in M. tuberculosis complex including human-type Mycobacterium tuberculosis and bovine-type Mycobacterium bovis.
• the M. tuberculosis complex is known to contain 10 to 12 copies of IS6110 which are commonly used in the PCR diagnosis of tuberculosis (Thierry, D., et al., Clin Microbiol. 1990;28(12):2668-2673).
• KENT et al. reported that IS6110 shows a difference in the false-positive risk, depending on a selection position of the primer (J. Clin Microbiol 1995;33(9):2290-2293).
• the primer of SEQ DD NO: 1 and the primer of SEQ ID NO: 2 are primer base sequences capable of amplifying only an IS6110 gene of M tuberculosis complex and exhibit very high sensitivity of more than 97% and a positive predictive value of more than 99% for the IS6110 gene region of the M tuberculosis complex, thus confirming extremely low risk of false positives. To the best of our knowledge, there is yet no report of such primers with high sensitivity and positive predictive value.
• the rpoB gene is in common among the Mycobacterium genus and it is therefore known that the presence of rpoB can be used for identification of mycobacterial species (Lee, Hye- Young, et al., Korean Patent Application
• the inventors of the present invention intend to employ an analysis method that is capable of rapidly and easily performing real-time PCR with high sensitivity for the Mycobacterium genus.
• the present inventors developed primers forming an amplification product having a size suited for the real-time PCR. That is, the primer of SEQ ID NO: 3 and the primer of SEQ ID NO: 4 are primers for targeting of the Mycobacterium genus-specific gene rpoB and form PCR amplification products having a length of lOObp or less.
• use of these primers results in a highly reliable, rapid, and accurate detection of Mycobacterium genus.
• composition for detection of M tuberculosis complex or Mycobacterium genus in accordance with the present invention may further comprise an internal control primer. Incorporation of the internal control is intended to confirm problems of false negatives, Ie. whether PCR was correctly carried out.
• a suitable gene may be optionally selected which is normally expressed irrespective of the presence/absence of M. tuberculosis or other mycobacterial species in the sample of interest.
• the aforesaid internal control primer may be a plant-derived gene-specific primer that is added to all the test samples.
• the plant-derived gene may be preferably a lectin gene.
• the lectin gene-specific primer may be a primer containing a base sequence as set forth in SEQ ID NO: 28 or a primer containing a base sequence as set forth in SEQ ID NO: 29.
• Table 1 below shows base sequences for the primers of SEQ DD NOs: 1 to 4, 28 and 29.
• the present invention provides a composition for detection of M. tuberculosis complex comprising a sense primer containing a base sequence as set forth in SEQ DD NO: 1 and/or an antisense primer containing a base sequence as set forth in SEQ ID NO: 2, as novel M. tuberculosis complex IS6110-specific primers.
• the present invention provides a composition for detection of Mycobacterium genus comprising a sense primer containing a base sequence as set forth in SEQ DD NO: 3 and/or an antisense primer containing a base sequence as set forth in SEQ DD NO: 4, as novel mycobacterial rpoB-specific primers.
• the mycobacterial rpoB-specific primers form PCR amplification products having a length of less than 1 OObp suitable for real-time PCR.
• the mycobacterial species may be M. tuberculosis or M bovis.
• the detection reliability of tuberculosis bacteria can be further improved by co-analysis of the rpoB gene in combination with the IS6110 gene of the M. tuberculosis complex.
• the mycobacterial strain may be nontuberculous mycobacteria (NTM).
• NTM nontuberculous mycobacteria
• the present inventors partially analyzed base sequences of rpoB genes of the following mycobacterial strains: M. acapulsewis, M. flavescens, M. gastin, M intracellulare, M. kansasii, M. pulveris, M. simiae, M. xenopi Schwabacher, M. austroafricamm, M. celatum, M. gastri, M. gordonae, M. agri, Masiaticum, M. cekatum, M. diernhoferi, M.fortuitum, M. nonchromogenicum, Mphlei, and M. genavense.
• ID Nos: 3 and 4 are effective for identification of the following 42 mycobacterial species and exhibit specificity for them.
• the present invention provides a composition for detection of M. tuberculosis complex or Mycobacterium genus, comprising at least one probe selected from the group consisting of: a probe containing a base sequence as set forth in SEQ ID NO: 5; a probe containing a base sequence as set forth in SEQ ID NO: 6; and a probe containing a base sequence as set forth in SEQ ID NO: 7.
• the composition may further comprise an internal control probe.
• the internal control probe is preferably a probe specific for a lectin gene that is a plant-derived gene.
• the internal control probe may be a probe containing a base sequence as set forth in SEQ ID NO: 30.
• the term "a probe containing a base sequence as set forth in SEQ ID NO: x" is intended to encompass a base sequence with a sequence homology of 95% or more.
• "a probe containing the base sequence of SEQ ID NO: x” is simply designated as "a probe of SEQ ID NO: x.
• the probe of SEQ ID NO: 5 specifically reacts with the IS6110 gene region, and the probes of SEQ ID NOs: 6 and 7 are probes that react in common with the rpoB gene of any member of the Mycobacterium genus that can be detected.
• the IS6110- or rpoB-specific probes in accordance with the present invention can be usefully used in PCR quantitative analysis by TaqMan assay or molecular beacon assay.
• the aforesaid probe is labeled with a fluorescent dye at 5' and 3' ends thereof.
• the 5-labeled fluorescent dye (reporter) and the 3-labeled fluorescent dye (quencher) may be ones exhibiting mutual interference therebetween. Accordingly, when the probe is combined with the IS6110 or rpoB gene in the sample, color development of the probe is limited. Then, when the probe is decomposed with PCR, the 3-labeled fluorescent dye is quenched whereas the 5-fluorescent dye undergoes color development. There is no particular limit to the 5'-labeled fluorescent dye.
• Examples of the 5'-labeled fluorescent dye may include, but are not limited to, 6-carboxyfluorescein (FAM), hexachloro-6- carboxyfluorescein (HEX), tetrachloro- ⁇ -carboxyfluorescein, and Cyanine-5 (Cy5).
• FAM 6-carboxyfluorescein
• HEX hexachloro-6- carboxyfluorescein
• Cyanine-5 Cyanine-5
• examples of the 3'-labeled fluorescent dye may include, but are not limited to, 6 ⁇ box ⁇ e1rarnethyl-rhodamine (TAMRA) and black hole quencher- 1,2,3 (BHQ-1,2,3).
• TAMRA 6 ⁇ box ⁇ e1rarnethyl-rhodamine
• BHQ-1,2,3 black hole quencher- 1,2,3
• Table 2 below shows base sequences for the probes of SEQ ID Nos: 5 to 7, and 30.
• composition and Kit for real-time PCR assay The present invention further provides a composition for analysis of M. tuberculosis complex and/or Mycobacterium genus by real-time PCR, comprising:
• At least one primer selected from the group consisting of: a primer containing a base sequence as set forth in SEQ ID NO: 1 ; a primer containing a base sequence as set forth in SEQ ID NO: 2; a primer containing a base sequence as set forth in SEQ ID NO: 3 ; and a primer containing a base sequence as set forth in SEQ ID NO: 4; and
• At least one probe selected from the group consisting of: a probe containing a base sequence as set forth in SEQ ID NO: 5; a probe containing a base sequence as set forth in SEQ ID NO: 6; and a probe containing a base sequence as set forth in SEQ ID NO: 7.
• composition may further comprise an internal control primer and an internal control probe to prevent false negatives.
• the internal control primer may be a sense primer containing a base sequence as set forth in SEQ ID NO: 28 or an antisense primer containing a base sequence as set forth in SEQ ID NO: 29.
• the internal control probe may be a probe containing a base sequence as set forth in SEQ ID NO: 30.
• the quantitative analysis composition in accordance with the present invention comprises the primer(s) for detection of tuberculosis bacteria or nontuberculous mycobacteria, the prob ⁇ ) for detection of tuberculosis bacteria or nontuberculous mycobacteria, and optionally, the internal control primer and probe.
• the real-time PCR assay can be carried out involving simultaneous amplification of two or three genes in a single tube in conjunction with DNA molecules extracted from a clinical sample, and real-time analysis and detection of the resulting amplification products.
• the composition may comprise:
• an M. tuberculosis complex IS6110-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 1 and the antisense primer containing a base sequence of SEQ ID NO: 2, and the probe containing a base sequence of SEQ ID NO: 5;
• a Mycobacterium genus rpoB-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 3 and the antisense primer containing a base sequence of SEQ ID NO: 4; and the probe containing a base sequence of SEQ ID NO: 6 and/or the probe containing a base sequence of SEQ ID NO: 7;
• an internal control-specific primer and probe mixture comprising the sense primer containing a base sequence of SEQ ID NO: 28 and the antisense primer containing a base sequence of SEQ ID NO: 29; and the probe containing a base sequence of SEQ ID NO: 30;
• the analysis composition of the present invention is comprised of three pairs of primers and three probes.
• the present inventors carried out multiplex real-time PCR As a result, it was confirmed that tuberculosis bacteria and other mycobacterial strains in the analyte sample can be detected and diagnosed with substantially complete exclusion of the risk of false positivity.
• the mixture (i) is capable of amplifying only the M. tuberculosis complex and includes a primer having high sensitivity of more than 97% and a positive predictive value of more than 99% for the IS6110 gene region and a probe that specifically binds to the IS6110 gene region which was amplified using the aforesaid primer.
• the mixture (ii) is capable of amplifying the rpoB gene that is a Mycobacterium genus-specific gene and includes a primer forming an rpoB gene region having a length suitable for real-time PCR-based assay and a probe that specifically binds to the rpoB gene region which was amplified using the aforesaid primer.
• the aforesaid probe binds to the rpoB gene of any member of the Mycobacterium genus that is detectable, whereby it is possible to detect the presence/absence of mycobacterial species through binding of the probe to the rpoB gene.
• composition may further comprise one or more probes containing an rpoB gene region having a different base sequence extracted from different mycobacterial species.
• mycobacterial species corresponding to the additionally incorporated probe can be identified.
• the composition of the present invention can be widely used for accurate diagnosis of tuberculosis infection.
• the real-time PCR assay may be carried out using commercially available real-time PCR equipment. Examples of the real-time PCR equipment may include, but are not limited to, SLAN real-time PCR detection system (LG Life Sciences, Korea), LightCyclerTM (Roche, Germany), ABI
• PRISMTM 7000/7700 (Applied Biosystems, USA), iCyclerTM (Bio-Rad, USA), Rotor-Gene " TM (Corbett, Australia), and OpticonTM (PharmaTech, USA).
• the analysis composition in accordance with the present invention may be used in qualitative analysis to confirm the presence/absence of M tuberculosis complex or Mycobacterium genus, or otherwise may be used in quantitative analysis of M tuberculosis complex or Mycobacterium genus. That is, the present invention provides a method for quantitative or qualitative analysis of tuberculosis bacteria or nontuberculous mycobacteria, using the aforesaid analysis composition.
• the qualitative analysis intended for identification of the presence/absence of M. tuberculosis complex or Mycobacterium genus can be carried out by confirming a time point at which a peak is observed by means of real-time PCR using the analysis composition.
• the quantitative analysis can be carried out by comparison with a standard curve.
• the quantitative analysis can be carried out according to the following procedure steps using the analysis composition: (1) mixing the analysis composition with bacterial DNA extracted from a sample of interest to prepare a gene analysis sample;
• Step (2) subjecting the gene analysis sample of Step (1) to real-time PCR to obtain a PCR product
• the sample-extracted bacterial DNA is added to the quantitative analysis composition comprising three primer pairs and three probes including an M. tuberculosis complex IS6110-specific primer pair and probe, a mycobacterium genus rpoB-specific primer pair and probe, and an internal control-specific primer pair and probe, and real time PCR is then carried out.
• each of IS6110-, rpoB- and internal control (lectin gene)-specific primer pairs and probes is attached to DNA molecules to result in amplification of a PCR product.
• the IS6110, rpoB and internal control-specific genes can be quantified by r ⁇ nfirming amplification of the genes through the fluorescence emitted upon decomposition of the probes attached to the IS6110, rpoB and lectin genes.
• the present invention provides an rpoB-specific nucleic acid of the Mycobacterium genus, the nucleic acid comprising at least one selected from the group consisting of base sequences of the following SEQ ID NOs: 8 to 27:
• M acapulsensis-spe ⁇ Sc nucleic acid containing a base sequence as set forth in SEQ ID NO: 8;
• nucleic acids contain the rpoB gene. Using these base sequences, it is possible to construct primers or probes that react in common with the Mycobacterium genus or it is possible to easily construct primers or probes having specificity corresponding to each mycobacterial species.
• Example 1 Construction of primers and probes for multiplex real-time PCR
• IS6110-specific primers and probes used herein were primer and probe sequences capable of amplifying only the M tuberculosis complex by analyzing the DNA sequence, deposited under accession number NC000962 in GenBank (www.ncbi.nlm.nih.gov') managed by the National Center for Biotechnology Information (NCBI), National Institutes of Health (NIH), using DNAsis which is a gene analysis program available from Hitachi Software, picking the appropriate sequence, and then analyzing the DNA sequence again with BLAST (www.ncbi.nlm.nih.gov/BLAST ⁇ .
• rpoB-specific primers and probes used herein were primer and probe sequences capable of amplifying only the Mycobacterium genus, by obtaining a
• Mycobacterium rpoB gene from the GenBank/Entrez Protein database, analyzing a conserved region of the rpoB gene using the software Clustal-X (see FIG. 1), picking the appropriate sequence, and then analyzing the DNA sequence again with BLAST (www.ncbi.nlm.nih.gov/BLAST/).
• sequenced primers are also capable of amplifying genes of the standard strains which were newly obtained.
• Example 1 The primers analyzed in Example 1 were synthesized on request by Metabion (Germany) according to the method such as "Synthesis of Oligonucleotide” described in a paragraph 10.42 of Molecular Cloning 3 rd ed (Sambrook and Russell, Cold Spring Harbor Laboratory Press, New York, USA, 2001).
• the precipitate was transferred to a 1.5 mL tube and 1 mL of a PBS buffer was added thereto. The resulting mixture was stirred and centrifuged at 13,000 rpm for 5 minutes. The supernatant was again discarded. 50 to 100 ⁇ i of 5% (w/v) Chelex 100 resin (Bio- Rad) was added to the precipitate, and the resulting mixture was heated at 100 ° C for 20 minutes and centrifuged at 13,000 rpm for 3 minutes. Supernatant with extracted DNA was used as template for PCR amplification.
• Example 4 Multiplex real-time PCR assay using primers and probes (1) PCR compositions were prepared according to a composition formula given in Table 3 below, and PCR was carried out in a SLAN real-time PCR detection system (LG Life Sciences, Korea) under the reaction conditions set forth in Table 4 below.
• reaction products were determined on a real-time basis. After the reaction was complete, the results were analyzed using a SLAN 7.0 program.
• FIG.2 M. tuberculosis complex-positive
• FIG. 3 Mycobacterium genus-positive
• FIG.4 Negative.
• Example 6 Diagnostic comparison of conventional culture method (MGIT, BD, USA) and Inventive kit for M. tuberculosis complex and Mycobacterium genus
• M. tuberculosis complex and Mycobacterium genus was compared between a conventional culture method (MGIT, BD, USA) and a kit of the present invention.
• the comparative culture method was carried out according to the manufacturer's instructions.
• the mycobacterial species was confirmed with an amplicor MTB kit (Roche Diagnostic Systems, NJ, USA) and base sequence analysis. The results obtained are given in Table 5 below.
• sensitivity refers to the proportion of people with disease in whom the test result is positive.
• specificity refers to the proportion of people without disease who have a negative test result.
• positive predictive value refers to the probability of the disease being present, among those with positive diagnostic test results.
• negative predictive value refers to the probability that the disease was absent, among those whose diagnostic test results were negative.
• Example 3 The samples obtained in Example 3 were respectively subjected to AFB staining which has been conventionally used in the confirmation of nontuberculous mycobacteria (NTM) and RT- PCR using a kit of the present invention. For comparison, the results obtained are given in Table 6 below.
• (+) represents NTM-negative
• (+) represents NTM-positive
• an increasing number of (+) represents higher concentration of NTM.
• AFB staining were also NTM-positive in RT-PCR results obtained according to the present invention.
• Example 8 Inventive RT-PCR assay for various mycobacterial species
• a value of less than 35 represents "Mycobacterium-iposii ⁇ yQ" and IC represents "internal control”.
• Q represents a threshold cycle number, Ie. the number of cycles that pass through a critical value when RT-PCR was performed, and therefore No Q represents that there is no threshold cycle.

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