CN110168109B - Diagnostic method for detecting and identifying tubercle bacillus and non-tubercle bacillus infection, confirmation of drug resistance of tubercle bacillus to rivastigmine and reagent group thereof - Google Patents
Diagnostic method for detecting and identifying tubercle bacillus and non-tubercle bacillus infection, confirmation of drug resistance of tubercle bacillus to rivastigmine and reagent group thereof Download PDFInfo
- Publication number
- CN110168109B CN110168109B CN201780061679.7A CN201780061679A CN110168109B CN 110168109 B CN110168109 B CN 110168109B CN 201780061679 A CN201780061679 A CN 201780061679A CN 110168109 B CN110168109 B CN 110168109B
- Authority
- CN
- China
- Prior art keywords
- mycobacterium
- tuberculosis
- mycobacterium tuberculosis
- qmap
- tubercle bacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation 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
- 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
-
- 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
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B45/00—ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B5/00—ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
-
- 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
- C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
- C12Q2563/107—Nucleic acid detection characterized by the use of physical, structural and functional properties fluorescence
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application relates to a diagnostic method and a reagent group for simultaneously detecting and identifying mycobacterium tuberculosis and nontuberculous mycobacterium and judging rifampicin and isoniazid resistance of the mycobacterium tuberculosis based on a quantaMatrix measuring platform.
Description
Technical Field
The application relates to a diagnostic method and a reagent group for simultaneously detecting and identifying mycobacterium tuberculosis and nontuberculous mycobacterium and judging rifampicin resistance of the mycobacterium tuberculosis based on a quantaMatrix measuring platform.
Background
Tuberculosis is a chronic infectious disease caused by Mycobacterium Tuberculosis (MTB), and its prevalence rate is steadily decreasing in korea, but is still higher than in other countries. Despite the use of effective antitubercular drugs, incurable tuberculosis, such as multidrug-resistant tuberculosis and drug-resistant tuberculosis, has increased. About 800 million people worldwide suffer from tuberculosis every year, and about 200 million people die of the disease every year. Therefore, rapid judgment of drug resistance of the refractory mycobacterium tuberculosis is essential for effective treatment and survival of patients. Tuberculosis is also caused by nontuberculous mycobacteria (NTM). In countries other than Korea, especially in areas where nontuberculous mycobacteria are frequently found, 30% to 50% of sputum samples are smear-positive for nontuberculous mycobacteria. It is reported that the number of tuberculosis patients caused by nontuberculous mycobacteria is increasing with the development of each country. Therefore, mycobacterial identification is very important [ Wright PW, Wallace RJ Jr, Wright NW, Brown BA, Griffith DE. J Clin Microbiol 1998; 36: 1046-9; marras TK and Daley cl. clin Chest Med 2002; 23: 553-67; diagnosis and treatment of diseases caused by nontuberculous mycobacteria. This is an official statement of the american thoracic association. Management of pathogenic mycobacterial infections: 1999 Joint tuberculosis Committee guidelines, Shorax 2000; 55:210-8].
It is known that the incidence of diseases caused by Korean non-tuberculous mycobacteria is low. In this case, most patients with a positive mycobacterial smear are diagnosed with tuberculosis and are usually treated with antitubercular drugs. Non-tuberculous mycobacteria were reported to be detected in 10.3-12.2% mycobacterial smear positive sputum samples in Korea, and this ratio was increased to 30% [ Koh WJ, Kwon OJ, Yu CM, Jeon K, Suh GY, Chung MP, et al Tuberc Respir Dis 2003; 54:22-32]. In addition, non-tuberculous mycobacteria can cause disease in immunosuppressed patients and are difficult to diagnose. Since natural drug resistance of nontuberculous mycobacteria varies depending on the species thereof, the treatment method for tuberculosis caused by nontuberculous mycobacteria may differ from the treatment method for tuberculosis caused by tuberculous mycobacteria [ Koh WJ, Kwon OJ, Lee ks.j Korean Med Sci 2005; 20: 913-25; wagner D and Young LS; nontuberculous mycobacterial infection: clinical reviews. Infection 2004; 32:257-70].
Therefore, there is a need for a suitable test for rapidly differentiating between Mycobacterium tuberculosis and nontuberculous mycobacteria.
Mycobacterial smear and culture tests are commonly used for tuberculosis diagnosis. The mycobacterial smear test is advantageous in that results can be obtained quickly and the method is easy to carry out, but has disadvantages of poor sensitivity and specificity and difficulty in distinguishing between mycobacterium tuberculosis and nontuberculous mycobacteria. Culture tests are the most accurate diagnostic criteria for tuberculosis. However, the culture test has a disadvantage in that the slow growth of mycobacterium tuberculosis takes a long time to diagnose tuberculosis, and the long culture results in frequent contamination of other bacteria [ Yang HY, Lee HJ, Park WY, Lee KK, Suh jt. korean J Lab Med 2006; 26:174-8]. Automated testing methods using liquid media have recently emerged to reduce the time required for mycobacterium tuberculosis culture. However, these methods also require additional identification procedures because they cannot distinguish between mycobacterium tuberculosis and nontuberculous mycobacteria [ Yang HY, Lee HJ, Park WY, Lee KK, Suh jt. korean J Lab Med 2006; 26:174-8]. The microorganism-based diagnosis is used as the gold standard for judging drug resistance. According to these methods, drug resistance is determined by culturing Mycobacterium tuberculosis in a sputum sample of a patient, followed by culture in a medium containing a drug. The procedure takes at least 4-8 weeks to determine whether the M.tuberculosis is drug resistant.
Delayed tuberculosis treatment by drug resistant mycobacterium tuberculosis results in local spread of drug resistant mycobacterium tuberculosis infection. Therefore, it is very important to judge the drug resistance of mycobacterium tuberculosis at the time of initial tuberculosis diagnosis. For this reason, molecular diagnosis is required to rapidly judge drug resistance.
Recently, many tuberculosis diagnoses based on advanced molecular diagnostic techniques have been developed [ Chakravorty S and Tyagi JS. A novel multifunctional method for detecting mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture and PCR. J Clin Microbiol 2005; 43: 2697-702; kim YJ, Park MY, Kim SY, Cho SA, Hwang SH, Kim HH, et al. 28:34-8]. Stable diagnosis of tuberculosis is based on evidence of mycobacterium tuberculosis infection. Therefore, direct detection of MTB in clinical samples is considered to be the most reliable method.
Under such circumstances, methods for detecting Mycobacterium tuberculosis by directly isolating them from patients with high sensitivity and specificity based on rapid molecular biology techniques are increasingly used. Such molecular biology techniques include, for example, PCR [ Yang HY, Lee HJ, Park WY, Lee KK, Suh JT. Korean J Lab Med 2006; 26: 174-8; chakravorty S and Tyagi JS.J Clin Microbiol 2005; 2697-; 28: 34-8; jung CL, Kim MY, Seo DC, Lee ma. korea J Clin Microbiol 2008; 1; 29-33], hybrid [ Makinen J, Marjamaki M, Marttila H, Soini H. Clin Microbiol Infect.2006; 12: 481-3; padilla E, Gonzalez V, manterala JM, Perez a, quelada MD, Gordillo S, et al.j Clin microbiological.2004; 42: 3083-8; sanguinetitti M, B Posteraro, F Ardito, S Zaneti, A Cinglani, L Sechi, et al.J. Clin Microbiol.1998; 36: 1530-3; tortuli, E, A Mariottini and G Mazzarelli. J Clin Microbiol. 2003; 4418-20] and oligonucleotide arrays [ Park H, Jang H, Song E, Chang CL, Lee M, Jeong S, et al.J. Clin Microbinary 2005; 43:1782-8]. One of the most widely used methods for identifying non-tubercular Mycobacteria is PCR-restriction fragment length polymorphism (PCR-RFLP) analysis (PRA). PRA can be carried out on the gene regions of Mycobacterium tuberculosis and nontuberculous mycobacteria by PCR amplification, the amplified gene regions are treated by proper restriction enzymes, and bacteria [ Lee HY, Park HJ, Cho SN, Bai GH, Kim SJ.J. Clin Microbiol.2000 are identified according to the characteristics of fragments; 2966-71; bannalikar AS, Verma r.indian J Med res.2006; 123: 165-72; aravinhan V, Sulochana S, Narayanan S, Paramasivam CN, Narayanan PR.Indian J Med Res.2007; 126:575-9].
However, in the case where two or more species of bacteria (including Mycobacterium tuberculosis and nontuberculous mycobacteria) coexist, the characteristics of the fragment after treatment with the restriction enzyme are complicated, making it impossible to identify the species of bacteria. To eliminate this inconvenience, molecular diagnostic tests based on the Quanta Matrix Assay Platform (QMAP) have recently been developed for simultaneous diagnosis and identification of mycobacterium tuberculosis and nontuberculous mycobacteria, and even mycobacterium tuberculosis and nontuberculous mycobacteria from a mixture of various bacterial species. QMAP based on the suspended array technology was originally developed by quanta matrix corporation (see korean patent No. 1011013100000(2011, 12/26 days) and patent No. 1015823840000(2015, 12/28 days).) according to the QMAP test, probes were combined with magnetic microdisks having a size of 50 μm, and the microdisks combined with the probes were reacted with PCR products, and mutation points were determined by fluorescence.
Disclosure of Invention
Technical problem to be solved
The present application addresses the above needs and it is an object of the present application to provide a novel diagnostic method for the detection and identification of mycobacterium tuberculosis and nontuberculous mycobacteria and the judgment of rifampin resistance to mycobacterium tuberculosis.
It is another object of the present application to provide a novel diagnostic reagent set for detecting and identifying Mycobacterium tuberculosis and nontuberculous mycobacteria, and judging rifampin (rifampin) resistance of Mycobacterium tuberculosis.
Technical scheme for solving technical problem
In one aspect, the present application provides a method for simultaneously detecting and identifying mycobacterium tuberculosis and nontuberculous mycobacteria, and a method for determining rifampin (rifampin) and isoniazid (isoniazid) resistance of mycobacterium tuberculosis, the method comprising a) isolating DNA from a sample, b) amplifying the DNA by PCR using primers having sequences shown in sequence numbers 1 to 12, and c) hybridizing the PCR amplification product to a disc coupled with an oligomer probe having a sequence shown in sequence numbers 13 to 68, and imaging the disc by quanta matrix assay platform software.
In one embodiment of the present application, the Mycobacterium tuberculosis and nontuberculous Mycobacterium strains are preferably selected from Mycobacterium tuberculosis (Mycobacterium tuberculosis) H37Rv, Mycobacterium avium (m.avium), Mycobacterium intracellulare (m.intercellulare), Mycobacterium scrofulum (m.scrofuleruleum), Mycobacterium abscessus (m.absessus), Mycobacterium chelonae (m.chelonae), Mycobacterium fortuitum (m.foritum), Mycobacterium ulcerosa (m.ulcerans), Mycobacterium marinum (m.marinaum), Mycobacterium kansasii (m.kanssoensis), Mycobacterium nipponensis (m.geneveense), Mycobacterium simian (m.simiaria), Mycobacterium terrestris (m.terrestris), Mycobacterium incognitum (m.aestivum), Mycobacterium tuberculosis Mycobacterium bufonii (m.xenopi), mycobacterium exotica (m.peregrinum), and mycobacterium flavum (m.flavescens), but is not limited thereto.
In another embodiment of the present application, the primer is preferably biotinylated, but the present application is not limited thereto.
The present application also provides a reagent set for simultaneous detection and identification of mycobacterium tuberculosis and nontuberculous mycobacteria and determination of rifampicin and isoniazid resistance of mycobacterium tuberculosis, the reagent set comprising primers having sequences shown in seq id nos 1-12 and disks conjugated with oligomer probes having sequences shown in seq id nos 13-68.
The reagent set preferably further includes DNA polymerase, dNTP and buffer as reagents for PCR amplification, but the reagents are not limited thereto.
The present application will now be described.
In the present application, the validity of QMAP-based dual-ID developed by the present inventors has been evaluated in solid and liquid media.
The diagnostic method of the present application (hereinafter also referred to as "QMAP-based Myco-ID") is a molecular diagnostic test method capable of detecting and identifying 26 species in total (22 and 3 groups) including mycobacterium tuberculosis and major non-mycobacterium tuberculosis and judgment of rifampicin resistance of mycobacterium tuberculosis. The major nontuberculous mycobacteria are M.avium, M.intracellulare, M.scrofula, M.abscessus complex, M.chelona, M.fortuitum complex, M.ulcerans/M.marinum, M.kansasii, M.geneveavense/M.simiae, M.simulae, M.terreus, M.xenogenium, M.trichoderma, M.inochromogenes, hidden mycobacteria (m.celatum), gordon mycobacteria (m.gordonae), chitsugami (m.szulgail), myxoma mycobacteria (m.mucogenium), umbellier mycobacteria (m.aubagnense), marmor mycobacteria (m.malmoense), mycobacterium phlei vaccine (m.phlei), mycobacterium smegmatis (m.smegmatis), mycobacterium bufonis (m.xenopi), exotic mycobacteria (m.peregrinum)/septicemia mycobacteria (m.septicium), and mycobacterium flavum (m.flavescens). Specifically, the diagnostic method of the present application amplifies an rpoB gene region containing a species-specific polymorphism by using a biotinylated primer (Korean patent No.10-1377070 (3/12 2014)) and reacting the resulting PCR product with a micro-magnetic disk to which a species-specific probe is attached. The Myco-ID based QMAP can be done automatically, helping to save time. Specifically, QMAP-based Myco-ID automatically tested in 96-well plates required a total of 3 hours (including 30 minutes for DNA extraction, 1 hour for PCR, and one and a half hours for TB/NTM detection and identification and rifampicin resistance determination).
Advantageous effects
The QMAP-based Myco-ID of the present application can accurately identify pathogenic bacteria of tuberculosis in a short time because it can distinguish mycobacterium tuberculosis from nontuberculous mycobacteria, identify nontuberculous mycobacteria, and determine rifampin and isoniazid resistance of mycobacterium tuberculosis. Thus, the QMAP-based Myco-ID of the present application provides useful information for providing a correct drug prescription for non-tubercular Mycobacteria. It is believed that the QMAP-based Myco-ID of the present application will reduce the risk of misdiagnosis of mycobacterium tuberculosis versus nontuberculous mycobacteria when mycobacterium tuberculosis is mixed with fast growing nontuberculous mycobacteria during liquid culture increased use. Therefore, the QMAP-based Myco-ID of the present application is very useful in diagnosing infectious diseases caused by Mycobacterium tuberculosis and nontuberculous mycobacteria.
Drawings
Fig. 1 is a diagram of a QMAP system.
FIG. 2 shows the results of REBA Myco-ID. Lanes 1-2, 9, 11, 13: m. intracellulare; lane 3: m. Mycobacteria mucosae; lanes 4-8, 16-19: mycobacterium tuberculosis, lane 10: mycobacterium malaysiae (m. masssyliense), lane 12: mycobacterium avium, lane 14: mycobacterium fortuitum; lane 15: mycobacterium abscessus, lane 20: and (5) negative control.
Detailed Description
The present application will be described in more detail with reference to the following non-limiting examples. However, these examples are for illustrative purposes only and should not be construed as limiting the scope of the present application.
Solid bacterial cultures (234 specimens) and liquid bacterial cultures (297 specimens) of mycobacterium tuberculosis suspected patients for hospitalization at Seoul Asan Medical Center (korea) and saint wensen Hospital (st. vincent's Hospital) (korea) were required for mycobacterial smear and culture trials during the period of 2009 from 5 months to 2009 9 months.
Mycobacterial smear and culture tests for detection of mycobacterium tuberculosis were performed in the seoul dental center (korea) and the san venson hospital (korea). Mycobacterial smear tests were performed by zier-nielsen (Ziehl-Neelsen) staining using phenol fuchsin (Carbol-fuchsin) and the results were recorded according to standardized procedures employed by the american centers for disease control and prevention. The culture test was performed as follows. First, each sample was treated with NaOH. The collected bacterial cells were then inoculated into 3% Ogawa medium (Asan Pharmaceuticals co., ltd., korea) and BACTEC MGIT 960(Becton Dickinson Microbiology System, Sparks, Md, usa) to culture pathogenic bacteria present in the sample.
Example 1: isolation and identification of Mycobacteria
Each sample was inoculated into 3% Ogawa medium and BACTEC MGIT 960 system and cultured at 37 ℃. A portion of the medium was collected, heated at 100 ℃ for 20 minutes, and centrifuged at 13,000rpm for 5 minutes. The collected supernatant was used for subsequent experiments. The MTB-ID PCR reagent set is used to distinguish Mycobacterium tuberculosis from nontuberculous mycobacteria by molecular diagnostic method.
Example 2: REBA Myco-ID
For REBA Myco-ID, the nucleic acid isolated from each sample was amplified by PCR using biotinylated primers. First, the nucleic acid was pre-denatured at 94 ℃ for 5 minutes. The reaction product was then denatured at 94 ℃ for 30 seconds, followed by 45 ℃ annealing and an extended cycle at 65 ℃ for 30 seconds. The final cycle was carried out at 72 ℃ for 7 minutes to obtain a 270-bp PCR product. The PCR product is amplified.
REBA Myco-ID was performed using the amplified PCR product. Experimental conditions were according to the manufacturer's experimental guidelines. The experimental procedure is as follows. The PCR product was mixed with the same amount of denaturing solution (0.2N NaOH, 0.2mM EDTA), left to stand at room temperature for 5 minutes, and a REBA Myco-ID strip (M & D, Korea) which had been prepared by previously diluting with 2X SSPE/0.1% SDS was added. The reaction was allowed to proceed at 50 ℃ for 30 minutes. The reaction mixture was washed twice with a Washing Solution (WS) at 62 ℃ for 10 minutes and diluted to 1: 2000(v/v) alkaline phosphatase-labeled streptavidin conjugate (Roche, Mannheim, Germany). The reaction was allowed to proceed for 30 minutes at room temperature. After completion of the reaction, the band was washed twice with TBS solution (pH7.5) for 1 min at room temperature and reacted with NBT/BCIP (nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, toluidine salt (Roche, Germany)) diluted to 1:50 with TBS solution (pH 9.5) for 5 min at room temperature. NBT/BCIP is a chromogenic substrate solution that reacts with alkaline phosphatase. Detecting the PCR product bound to the probe.
Example 3: dual ID based on QMAP
For QMAP-based Myco-ID, nucleic acids were isolated from each sample by PCR amplification using biotinylated primers. First, the nucleic acid was pre-denatured at 94 ℃ for 5 minutes. The reaction product was then denatured at 94 ℃ for 30 seconds, followed by 45 ℃ annealing and extended cycling at 65 ℃ for 30 seconds. The final cycle was carried out at 72 ℃ for 7 minutes to obtain a 250-bp PCR product. The PCR product is amplified.
The amplified PCR product was mixed with the same amount of denaturing solution (0.2N NaOH, 0.2mM EDTA), left to stand at room temperature for 5 minutes, and added to a coupled disk (Quantamatrix, Seal, Korea), which had been previously prepared by dilution with hybridization buffer. The reaction was allowed to proceed at 40 ℃ for 30 minutes. The reaction mixture was washed three times with a Washing Solution (WS) at 25 ℃ for 1 min and diluted to 1: 2000(v/v) of streptavidin-R-phycoerythrin conjugate (R-phycerythrin conjugate) (Prozyme, San Leandro, Calif.). The reaction was allowed to proceed for 10 minutes at room temperature. After the reaction was completed, the microplates were washed three times with the washing solution for 1 minute at room temperature and their images were photographed in an automatic manner using the provided QMAP software to detect MTB or NTM, isolate NTM, and judge rifampicin and isoniazid resistance of mycobacterium tuberculosis.
The sequences of the primers and probes used are described in table 8. The synthesis of primers and probes was requested from the Korean Bioneer company.
The results obtained in examples 1 to 3 are described below.
Differentiation of Mycobacterium tuberculosis from non-Mycobacterium tuberculosis after amplification of DNA isolated from culture samples by QMAP-based assay sensitivity and specificity multiplex PCR (MTB-ID)
To investigate the sensitivity of QMAP-based assays for MTB and NTM, each of the standard strains (Mycobacterium tuberculosis) H37Rv, Mycobacterium avium (m.aveum), Mycobacterium intracellulare (m.intercellulare), Mycobacterium scrofulum (m.scrofuleruleum), Mycobacterium abscessus (m.absceis), Mycobacterium chelonensis (m.chelona), Mycobacterium fortuitum (m.foritum), Mycobacterium oceanic (m.marina), Mycobacterium kansasii (m.kansasii), Mycobacterium japanese (m.geneva), Mycobacterium terrestris (m.terrestris), Mycobacterium ionogenes (m.leucogenium), Mycobacterium crypthecogenum (m.glauca), Mycobacterium gordonii (m.gordonae), Mycobacterium tsukii (m.brewskii), Mycobacterium smegmatis (m.parakugenum), Mycobacterium xenophilus (m.paragallinarum), Mycobacterium xenopus (m.sp.sp.), Mycobacterium tuberculosis (m.paragallinarum, Mycobacterium tuberculosis (m.sp.m.sp., Mycobacterium tuberculosis (m.sp.sp.m.m.sp., Mycobacterium tuberculosis (m.sp.m.sp., Mycobacterium xenopus. bacillus), Mycobacterium tuberculosis (m.sp., Mycobacterium xenopus. bacillus sp., Mycobacterium), Mycobacterium tuberculosis (m.sp., Mycobacterium xenopus. vaccine (m.10. and Mycobacterium tuberculosis (m.sp., Mycobacterium), Mycobacterium xenopus. before-bacillus (m.sp. vaccine (m.sp., Mycobacterium), Mycobacterium tuberculosis. sp., Mycobacterium (m.sp., Mycobacterium), Mycobacterium (m. sp., Mycobacterium tuberculosis, Mycobacterium (m. sp., Mycobacterium), Mycobacterium (m. sp., Mycobacterium (m. sp., Mycobacterium), Mycobacterium (m. sp., Mycobacterium), Mycobacterium (m. sp., Mycobacterium (m. sp., Mycobacterium), Mycobacterium (m. sp., Mycobacterium (m. sp., Mycobacterium), Mycobacterium (m. sp. 1ng, 100pg, 10pg, 1pg, 100fg and 10fg (1 bacillus)). As a result, the detection limit was 1pg (100 bacilli) to 100fg (10 bacilli) (Table 1). To judge QMAP-based specificity, a total of 108 strains (MTB H37Rv, 44 NTM strains and 63 non-mycobacterial strains) were used for the test. 28 standard strains were detected precisely on disks coupled with specific probes, and three probes were detected together with Mycobacterium fortuitum-M.marinum, M.kansasii-M.gastri and M.geneva-M.avium (Table 2).
TABLE 1 analytical sensitivity of QMAP System
Abbreviations: CV, coefficient of variation
TABLE 2 analytical specificity of QMAP System Using 180 Standard strains
Abbreviations: ATCC, American type culture Collection (American type culture Collection); CIP, Pasteur Institute of culture Collection (Collection Institute Pasteur); DML, department of University Biomedical and Laboratory Science clinical Microbiology Laboratory (Diagnostic Microbiology Laboratory, biological Laboratory Science, Yonsei University); KACC, Korean Agricultural culture collection (Korean Agricultural culture collection); KCMF, Korean Culture Collection Medical; UD, Undetermined (Undetermined)
Evaluation of the effectiveness of QMAP-based Myco-ID in clinical samples
To evaluate the effectiveness of QMAP-based Myco-ID, the MTB/NTM detection and NTM identification of strain 531 (including 234 solid bacterial cultures and 297 liquid bacterial cultures) was tested. As a result, 234 solid bacterial cultures were identified as 223 MTB strains and 11 NTM strains, and the fluorescence intensities of the 223 MTB strains and the 11 NTM strains detected were in the ranges of 2074-4765 (average 4255.6 + -SD 458.7) and 609-3569 (1892 + -923.2, respectively). 212 MTB strains and 77 NTM strains in 297 liquid media showed positive signals in the QMAP test. Of the total 531 strains, 527 (99.2%) were accurately identified as MTB and NTM strains. Two of the 4 strains not detected were negative for the culture, and the other two strains were identified by sequencing as Rhodococcus erythropolis and Rhodococcus johnsonii (R.jostii). The results of QMAP-based Myco-ID were again confirmed by comparison with the results of another molecular diagnostic method (REBA Myco-ID) (FIG. 2). The results of QMAP-based Myco-ID were found to be in full agreement with those of REBA Myco-ID (Table 3).
TABLE 3 comparison of conventional methods with QMAP System and REBA Myco-ID for MTB and NTM detection after DNA isolated from 531 total bacterial cultures was amplified
Abbreviations: AFB, acid fast bacilli; MTB, mycobacterium tuberculosis; NTM, nontuberculous mycobacteria; ND, not detected
a No two NTM strains were detected in the QMAP system and REBA Myco-ID, and Rhodococcus erythropolis and Rhodococcus johnsonii were identified by rpoB sequencing.
b mixed cases of NTM and NTM
c two smear/culture negative cases
Detection profile of nontuberculous mycobacteria
94 of a total of 531 cultured bacterial samples were detected as NTM species by the QMAP-based system. 94 strains of bacteria were distributed in the following order: 36 strains (39.1%), 21 strains (22.8%), 14 strains (15.2%) and 7 strains (7.6%) were identified as M.intracellulare, M.avium, M.abscessus complex and M.fortuitum, respectively. 4 strains (4.3%), 2 strains (2.2%) and 1 strain (1.1%) were identified as Mycobacterium gordonae, Mycobacterium kansasii and Mycobacterium cheloni, respectively. Two mixtures of mycobacterium avium and mycobacterium intracellulare (2.2%), two mixtures of mycobacterium avium and mycobacterium abscessus (2.2%), one mixture of mycobacterium avium and mycobacterium mucosae (1.1%), and one mixture of mycobacterium avium and mycobacterium mucosae (1.1%) were detected (table 4). These results are in good agreement with those of REBA Myco-ID. From the above results, it can be seen that a mixture of two or more kinds of nontuberculous mycobacteria can be isolated even by the QMAP system.
TABLE 4 comparison of the distribution of NTM species isolated by the REBA Myco-ID and QMAP systems
Evaluation of the effectiveness of rifampicin and isoniazid resistance by the QMAP System
435 out of the total 531 strains was detected as a Mycobacterium tuberculosis strain. Among 435 strains, 223 solid bacterial cultures whose results of conventional Drug Susceptibility Test (DST) are known were used to evaluate the effectiveness of rifampicin resistance determined by QMAP system. 27 of the 223 M.tuberculosis strains (12.1%) demonstrated rifampicin resistance, which is well matched with the traditional DST results. The drug resistant regions of 27 resistant strains were 531TTG (13, 48.1%),. DELTA.WT 4(5, 18.5%),. DELTA.WT 5 and 526TAC (3 cases each, 11.1%), and 516GTC,. DELTA.WT 4,. DELTA.WT 2 and. DELTA.WT 4 (1 case each, 3.7%), respectively. These results were compared to the results of REBA MTB-MDR for rifampin resistance determination. Rifampicin resistance and drug resistance regions determined by QMAP system were consistent with those determined by REBA MTB-MDR (table 5).
TABLE 5 comparison of conventional methods after amplification of DNA isolated from a total of 226 bacterial cultures with QMAP System and REBA Myco-ID for rifampicin resistance detection
108 strains were identified as having resistance to isoniazid. 82 of 108 strains (75.9%) had drug resistance in katG315 (table 6). These results were compared with the results of DNA sequencing for determination of isoniazid resistance. The isoniazid resistance and resistance regions determined by the QMAP system were consistent with those determined by REBA Myco-ID (table 6).
[ Table 6] comparison of conventional methods with QMAP System and DNA sequencing for isoniazid resistance detection after amplification of DNA isolated from bacterial cultures
Furthermore, the results of the QMAP system were compared with phenotypic DST results. The sensitivity to Rifampicin (Rifampicin) and Isoniazid (Isoniazid) resistance based on QMAP was 96.4% (106/110) and 75% (108/144), respectively. In contrast, sensitivity to rifampicin and isoniazid resistance based on QMAP was 100% (124/124; 95% CI: 0.9743-1.0000) and 100% (110/110, 95% CI: 0.9711) -1.0000), both compared to the DNA sequencing results (Table 7).
[ Table 7] comparison of the results of QMAP System with the DST results and DNA sequencing results
Abbreviations: DST, drug susceptibility test; PPV, positive predictive value; NPV, negative predictive value; CI, confidence interval.
[ Table 8] sequences of primers and probes used
Comparison of the methods of the present application with conventional REBA
As can be seen from FIG. 2, a total of 19 species including Mycobacterium tuberculosis were detected by REBA Myco-ID. In contrast, 7 additional bacterial species were isolated by the QMAP-based diagnostic method of the present application, as compared to by REBA Myco-ID. In summary, the QMAP system of the present application is capable of isolating, including Mycobacterium tuberculosis, M.marmoreus, M.phlei vaccines, M.smegmatis, M.bufonii, M.exoticus, M.septicum, and M.lutescens, as well as 26 major non-tuberculous mycobacterial species. (Mycobacterium avium, M.intracellulare, M.scrofulaceum, M.abscessus complex, M.chelonii, M.fortuitum, M.ulcerosa/M.oceanic, M.kansasii, M.henivalis/M.apeutic, M.terrae, M.chromogenes, M.crypticus, M.gordonii, M.chikungunyi, M.mucosae, M.onae).
REBA Myco-ID can only be used to distinguish between tuberculosis and NTM. In contrast, the QMAP-based diagnostic method of the present application can be used to rapidly judge the resistance to rifampicin, which is the most important primary anti-tubercular drug. Therefore, the use of the QMAP-based diagnostic method may be useful for screening tuberculosis therapeutic agents.
The QMAP-based molecular approach can be used to find drug-resistant sites for rifampicin and isoniazid sensitive and resistant tuberculosis (tables 5-7). The QMAP-based molecular approach of the present application shows better results than the conventional approach. The use of REBA Myco-ID is limited to the identification of Mycobacterium tuberculosis/NTM. Therefore, another molecular diagnostic method for tuberculosis (e.g., REBA MTB-MDR) is needed to perform additional tests to determine rifampin-resistance. In contrast, the method of the present application avoids the need for additional testing.
Another advantage of the QMAP based diagnostic method according to the present application is that the time required for the detection is reduced. REBA Myco-ID typically requires at least 5 hours to test in 96-well plates (only for REBA). In contrast, the QMAP-based diagnostic method can reduce the time required for detection by one hour and 40 minutes. Time saving is most important for diagnostics.
Claims (6)
1. A method for simultaneously detecting and identifying mycobacterium tuberculosis and nontuberculous mycobacteria and determining rifampin and isoniazid resistance of mycobacterium tuberculosis, the method comprising: a) isolating DNA from the sample, b) amplifying the DNA by PCR using primers having the sequences shown in SEQ ID Nos. 1 to 12, and c) hybridizing the PCR amplification products to disks coupled with oligo probes having the sequences shown in SEQ ID Nos. 13 to 68 and imaging the disks by QuantaMatrix assay platform software.
2. The method according to claim 1, wherein the Mycobacterium tuberculosis and nontuberculous mycobacteria strains are selected from the group consisting of Mycobacterium tuberculosis (M.tuberculosis) H37Rv, Mycobacterium avium (M.avium), Mycobacterium intracellulare (M.intercellulare), Mycobacterium scrofulae (M.Scrofulosum), Mycobacterium abscessus (M.absessus), Mycobacterium chelonii (M.chelonae), Mycobacterium fortuitum (M.fortuitum), Mycobacterium ulcerans (M.ulcerans), Mycobacterium marinum (M.marinum), Mycobacterium kansasii (M.kansasii), Mycobacterium geneva (M.avengense), Mycobacterium simian (M.simiae), Mycobacterium terrae (M.marrae), Mycobacterium chromatogenes (M.nocolosum), Mycobacterium not-producing (M.nosogenes), Mycobacterium crypthecogenum (M.celothecium), Mycobacterium gobenium (M.celothecium), Mycobacterium vaccae (M.gordongium), Mycobacterium tuberculosis (M.M.kuwanensis), Mycobacterium tuberculosis (M.M.M.kuwanesei), Mycobacterium tuberculosis (M.M.M.M.kuwanesei), Mycobacterium tuberculosis Mycobacterium smegmatis (m.smegmatis), mycobacterium bufonis (m.xenopi), mycobacterium exoticus (m.peregrinum), and mycobacterium flavum (m.flavescens).
3. The method of claim 1, wherein the primer is biotinylated.
4. A reagent set for simultaneously detecting and identifying mycobacterium tuberculosis and nontuberculous mycobacteria and judging rifampicin and isoniazid resistance of mycobacterium tuberculosis, characterized in that the reagent set comprises a primer having a sequence shown in sequence numbers 1 to 12 and a magnetic disk coupled with an oligomer probe having a sequence shown in sequence numbers 13 to 68.
5. The reagent set according to claim 4, wherein the Mycobacterium tuberculosis and the nontuberculous Mycobacterium strain are selected from the group consisting of Mycobacterium tuberculosis (M.tuberculosis) H37Rv, Mycobacterium avium (M.avium), Mycobacterium intracellulare (M.intercellulare), Mycobacterium scrofulae (M.scrofulaceae), Mycobacterium abscessus (M.abstiness), Mycobacterium chelonii (M.chelona), Mycobacterium fortuitum (M.fortuitum), Mycobacterium ulcerosa (M.ulcens), Mycobacterium marinum (M.marinum), Mycobacterium kansasii (M.kansasii), Mycobacterium genenii (M.geneveense), Mycobacterium simian (M.simiae), Mycobacterium terrestris (M.terrestris), Mycobacterium not producing (M.nosyneanum), Mycobacterium phlei (M.), Mycobacterium phlei (M.maceum), Mycobacterium phlegmarium, Mycobacterium phlei (M.gordonii) Mycobacterium smegmatis (m.smegmatis), mycobacterium bufonis (m.xenopi), exotic mycobacterium (m.peregrinum), and mycobacterium flavum (m.flavescens).
6. The reagent set of claim 4, wherein the primer is biotinylated.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20160099515 | 2016-08-04 | ||
| KR10-2016-0099515 | 2016-08-04 | ||
| PCT/KR2017/008168 WO2018026141A1 (en) | 2016-08-04 | 2017-07-28 | Diagnostic method for detection and identification of tuberculosis and non-tuberculosis mycobacteria and simultaneous confirmation of rifampin resistance of tuberculosis bacillus on basis of quanta matrix assay platform and kit therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110168109A CN110168109A (en) | 2019-08-23 |
| CN110168109B true CN110168109B (en) | 2022-09-20 |
Family
ID=61072945
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201780061679.7A Active CN110168109B (en) | 2016-08-04 | 2017-07-28 | Diagnostic method for detecting and identifying tubercle bacillus and non-tubercle bacillus infection, confirmation of drug resistance of tubercle bacillus to rivastigmine and reagent group thereof |
Country Status (4)
| Country | Link |
|---|---|
| KR (1) | KR102184637B1 (en) |
| CN (1) | CN110168109B (en) |
| RU (1) | RU2715591C1 (en) |
| WO (1) | WO2018026141A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102241251B1 (en) | 2019-07-26 | 2021-04-16 | 주식회사 수젠텍 | Multiplexing blot assay automation system |
| CN113136446A (en) * | 2021-06-02 | 2021-07-20 | 上海康黎诊断技术有限公司 | Method, primer group and kit for identifying non-tuberculous mycobacteria and detecting drug-resistant gene mutation |
| CN113360383A (en) * | 2021-06-09 | 2021-09-07 | 中电科思仪科技股份有限公司 | Automatic test software data dynamic synchronization method of comprehensive detection equipment |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006098753A (en) * | 2004-09-29 | 2006-04-13 | Toshiba Corp | Refractive index changing element and method |
| CN101434952A (en) * | 2007-10-16 | 2009-05-20 | 霍夫曼-拉罗奇有限公司 | Reagents and methods for detecting neisseria gonorrhoeae |
| CN102634575A (en) * | 2012-03-26 | 2012-08-15 | 中国人民解放军第三O九医院 | Rapid identification method and kit of novel mycobacterium strain |
| CN105308062A (en) * | 2013-02-20 | 2016-02-03 | 伊芙生物医学股份有限公司 | Methods and compositions for nanostructure-based nucleic acid sequencing |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU703947B2 (en) * | 1994-06-09 | 1999-04-01 | Innogenetics N.V. | Method for the detection of the antibiotic resistance spectrum of mycobacterium species |
| KR100446850B1 (en) * | 2001-07-19 | 2004-09-04 | 이혜영 | A method for identifying Micobacteria tuberculosis and non-tuberculosis Micobacteria, together with detecting resistance to an antituberculsis drug of Micobacteria obtained by mutation of rpoB gene |
| KR100454585B1 (en) * | 2001-10-09 | 2004-11-02 | 주식회사 에스제이하이테크 | Microarray comprising probes for Mycobacteria genotyping, M. tuberculosis strain differentiation and antibiotic-resistance detection |
| RU2376387C2 (en) * | 2005-12-26 | 2009-12-20 | Учреждение Российской академии наук Институт молекулярной биологии им. В.А. Энгельгардта РАН | Method for simultaneous detection of mycobacteria of tuberculosis complex and identification of mutations in dna of mycobacteria, which result in microorganisms resistance to rifampicin and isoniazid, on biological microchips, set of primers, biochip and set of oligonucleotide probes used in method |
| KR100990756B1 (en) * | 2008-05-22 | 2010-10-29 | 엠앤디 (주) | Probe for Detection of drug resistance of Mycobacterium tuberculosis and the kit |
| WO2011004957A2 (en) * | 2009-07-07 | 2011-01-13 | 엠앤디(주) | Probe for detecting the resistance of mycobacterium tuberculosis to drugs, and kit for same |
| KR101377070B1 (en) * | 2012-02-20 | 2014-03-20 | 엠앤디 (주) | Method for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria and composition therefor |
| KR101774995B1 (en) * | 2014-10-14 | 2017-09-05 | 주식회사 퀀타매트릭스 | Rapid antimicrobial susceptibility test using microbe cell morphological and growth change under different concentrations of various antimicrobial agents and automatied cell image analysis system using the same |
| RU2562866C1 (en) * | 2014-11-11 | 2015-09-10 | Федеральное Государственное Бюджетное Учреждение Науки Институт Молекулярной Биологии Им. В.А. Энгельгардта Российской Академии Наук (Имб Ран) | Detection method of dna of tuberculosis causative agent with simultaneous identification of its genotype and determination of genetic determinants of multiple and wide drug resistance, oligonucleotide microchip, set of primers and set of oligonucleotide probes, which are used in method |
-
2017
- 2017-07-28 WO PCT/KR2017/008168 patent/WO2018026141A1/en active Application Filing
- 2017-07-28 RU RU2019105616A patent/RU2715591C1/en active
- 2017-07-28 KR KR1020197003372A patent/KR102184637B1/en active IP Right Grant
- 2017-07-28 CN CN201780061679.7A patent/CN110168109B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006098753A (en) * | 2004-09-29 | 2006-04-13 | Toshiba Corp | Refractive index changing element and method |
| CN101434952A (en) * | 2007-10-16 | 2009-05-20 | 霍夫曼-拉罗奇有限公司 | Reagents and methods for detecting neisseria gonorrhoeae |
| CN102634575A (en) * | 2012-03-26 | 2012-08-15 | 中国人民解放军第三O九医院 | Rapid identification method and kit of novel mycobacterium strain |
| CN105308062A (en) * | 2013-02-20 | 2016-02-03 | 伊芙生物医学股份有限公司 | Methods and compositions for nanostructure-based nucleic acid sequencing |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20190022871A (en) | 2019-03-06 |
| RU2715591C1 (en) | 2020-03-02 |
| WO2018026141A1 (en) | 2018-02-08 |
| CN110168109A (en) | 2019-08-23 |
| KR102184637B1 (en) | 2020-11-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Palomino | Molecular detection, identification and drug resistance detection in Mycobacterium tuberculosis | |
| Somoskovi et al. | Laboratory diagnosis of nontuberculous mycobacteria | |
| Guo et al. | Rapid, accurate determination of multidrug resistance in M. tuberculosis isolates and sputum using a biochip system | |
| KR101458781B1 (en) | Composition for Detection of M. tuberculosis Complex and Mycobacteria Genus and Simultaneous Detection Method for M. tuberculosis Complex and Mycobacteria Genus with Multiplex Real Time PCR Using the Same | |
| Kox et al. | Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease | |
| KR101377070B1 (en) | Method for distinguishing between Mycobacterium tuberculosis and nontuberculous mycobacteria and composition therefor | |
| CN102634575A (en) | Rapid identification method and kit of novel mycobacterium strain | |
| KR20100031188A (en) | Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time pcr using the same | |
| CN110168109B (en) | Diagnostic method for detecting and identifying tubercle bacillus and non-tubercle bacillus infection, confirmation of drug resistance of tubercle bacillus to rivastigmine and reagent group thereof | |
| Lekhak et al. | Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis | |
| WO2010030049A1 (en) | Composition for detection of m. tuberculosis complex or mycobacteria genus and simultaneous detection method for m. tuberculosis complex and mycobacteria genus with multiplex real time pcr using the same | |
| Kalaiarasan et al. | Diagnostic performance of real time PCR and MALDI-TOF in the detection of nontuberculous mycobacteria from clinical isolates | |
| Foongladda et al. | Multi-probe real-time PCR identification of common Mycobacterium species in blood culture broth | |
| Fisher | Diagnosis of MDR-TB: a developing world problem on a developed world budget | |
| KR20030008620A (en) | A method for identifying Micobacteria tuberculosis and non-tuberculosis Micobacteria, together with detecting resistance to an antituberculsis drug of Micobacteria obtained by mutation of rpoB gene | |
| Kim et al. | Evaluation of dual-color fluorescence in situ hybridization with peptide nucleic acid probes for the detection of Mycobacterium tuberculosis and non-tuberculous mycobacteria in clinical specimens | |
| Stauffer et al. | Detection of Mycobacterium tuberculosis complex in clinical specimens by a commercial polymerase chain reaction kit | |
| EP2677040A1 (en) | Probe, chip, kit and method for detection of mycobacterium tuberculosis, non-tuberculous mycobacteria and drug resistant of mycobacterium tuberculosis | |
| KR102247643B1 (en) | A method for diagonising mycobacterium tuberculosis and non-tuberculosis mycobacterium in 2-step way and the kit thereof | |
| Oh et al. | Improved detection and differentiation of mycobacteria with combination of Mycobacterium Growth Indicator Tube and Roche COBAS AMPLICOR System in conjunction with Duplex PCR | |
| Wang et al. | Evaluation of the quantamatrix multiplexed assay platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria | |
| Glennon et al. | Detection and diagnosis of mycobacterial pathogens using PCR | |
| Palacios et al. | Comparison of solid and liquid culture media with the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples | |
| Johansen et al. | Rapid differentiation between clinically relevant mycobacteria in microscopy positive clinical specimens and mycobacterial isolates by line probe assay | |
| Bae et al. | Comparison of the Three Molecular Diagnostic Assays for Molecular Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria Species in Sputum Samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |