A Color-Reaction-Based Biochip Detection Assay for RIF and INH Resistance of Clinical Mycobacterial Specimens
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초록
The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.
Abstract
The widespread occurrence of drug-resistant Mycobacterium tuberculosis places importance on the detection of TB (tuberculosis) drug susceptibility. Conventional drug susceptibility testing (DST) is a lengthy process. We developed a rapid enzymatic color-reaction-based biochip assay. The process included asymmetric multiplex PCR/templex PCR, biochip hybridization, and an enzymatic color reaction, with specific software for data operating. Templex PCR (tem- PCR) was applied to avoid interference between different primers in conventional multiplex- PCR. We applied this assay to 276 clinical specimens (including 27 sputum, 4 alveolar lavage fluid, 2 pleural effusion, and 243 culture isolate specimens; 40 of the 276 were non-tuberculosis mycobacteria specimens and 236 were M. tuberculosis specimens). The testing process took 4.5 h. A sensitivity of 50 copies per PCR was achieved, while the sensitivity was 500 copies per PCR when tem-PCR was used. Allele sequences could be detected in mixed samples at a proportion of 10%. Detection results showed a concordance rate of 97.46% (230/236) in rifampicin resistance detection (sensitivity 95.40%, specificity 98.66%) and 96.19% (227/236) in isoniazid (sensitivity 93.59%, specificity 97.47%) detection with those of DST assay. Concordance rates of testing results for sputum, alveolar lavage fluid, and pleural effusion specimens were 100%. The assay provides a potential choice for TB diagnosis and treatment.
참고문헌 (31)
- Abebe G, "Tuberculosis drug resistance testing by molecular methods: opportunities and challenges in resource limited settings", J. Microbiol. Methods, 84, pp.155~160 (2011)
- Aziz MA, "Epidemiology of antituberculosis drug resistance (the global project on anti-tuberculosis drug resistance surveillance): an updated analysis", Lancet, 368, pp.2142~2154 (2006)
- Benson R, "Development and evaluation of a novel multiplex PCR technology for molecular differential detection of bacterial respiratory disease pathogens", J. Clin. Microbiol, 46, pp.2074~2077 (2008)
- Bobadilla-del-Valle M, "rpoB Gene mutations in rifampin-resistant Mycobacterium tuberculosis identified by polymerase chain reaction singlestranded conformational polymorphism", Emerg. Infect. Dis, 7, pp.1010~1013 (2001)
- Caoili JC, "Evaluation of the TB-Biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis", J. Clin. Microbiol, 44, pp.2378~2381 (2006)
- Chen Q, "Detection of C677T mutation of MTHFR in subject with coronary heart disease by hairpin probe with enzymatic color on microarray", Biosens. Bioelectron, 28, pp.84~90 (2011)
- Cherkasova E, "Microarray analysis of evolution of RNA viruses: evidence of circulation of virulent highly divergent vaccine-derived polioviruses", Proc. Natl. Acad. Sci. USA, 100, pp.9398~9403 (2003)
- Cruciani M, "Meta-analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without solid media, for detection of mycobacteria", J. Clin. Microbiol, 42, pp.2321~2325 (2004)
- Donnabella V, "Isolation of the gene for the beta subunit of RNA polymerase from rifampicin-resistant Mycobacterium tuberculosis and identification of new mutations", Am. J. Respir. Cell Mol. Biol, 11, pp.639~643 (1994)
- Edwards KJ, "Detection of rpoB mutations in Mycobacterium tuberculosis by biprobe analysis", J. Clin. Microbiol, 39, pp.3350~3352 (2001)
- Espasa M, "Direct detection in clinical samples of multiple gene mutations causing resistance of Mycobacterium tuberculosis to isoniazid and rifampicin using fluorogenic probes", J. Antimicrob. Chemother, 55, pp.860~865 (2005)
- Goble M, "Treatment of 171 patients with pulmonary tuberculosis resistant to isoniazid and rifampin", N. Engl. J. Med, 328, pp.527~532 (1993)
- Gryadunov D, "Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis", Clin. Microbiol. Infect, 11, pp.531~539 (2005)
- Guo Y, "Rapid, accurate determination of multidrug resistance in M. tuberculosis isolates and sputum using a biochip system", Int. J. Tuberc. Lung Dis, 13, pp.914~920 (2009)
- Han J, "Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology", J. Clin. Microbiol, 44, pp.4157~4162 (2006)
- Heep M, "Frequency of rpoB mutations inside and outside the cluster I region in rifampin-resistant clinical Mycobacterium tuberculosis isolates", J. Clin. Microbiol, 39, pp.107~110 (2001)
- Hemvani N, "A simple and economical in-house phage technique for the rapid detection of rifampin, isoniazid, ethambutol, streptomycin, and ciprofloxacin drug resistance in Mycobacterium tuberculosis, directly on decontaminated sputum samples", Int. J. Infect. Dis, 16, pp.e332~e336 (2012)
- Higgs BW, "Early detection of tuberculosis outbreaks among the San Francisco homeless: trade-offs between spatial resolution and temporal scale", PLoS One, 2, pp.e1284~ (2007)
- Khue PM, "A 10-year prospective surveillance of Mycobacterium tuberculosis drug resistance in France 1995-2004", Eur. Respir. J, 30, pp.937~944 (2004)
- Laurenzo D, "Mechanisms of drug resistance in Mycobacterium tuberculosis and current status of rapid molecular diagnostic testing", Acta Trop, 119, pp.5~10 (2011)
- Ling DI, "GenoType MTBDR assays for the diagnosis of multidrug-resistant tuberculosis:a meta-analysis", Eur. Respir. J, 32, pp.1165~1174 (2008)
- Morgan M, "A commercial line probe assay for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a systematic review and metaanalysis", BMC Infect. Dis, 5, pp.62~ (2005)
- Pang Y, "Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China", PLoS One, 7, pp.e32976~ (2012)
- Park H, "Comparison of a conventional antimicrobial susceptibility assay to an oligonucleotide chip system for detection of drug resistance in Mycobacterium tuberculosis isolates", J. Clin. Microbiol, 44, pp.1619~1624 (2006)
- Pholwat S, "Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis", J. Clin. Microbiol, 50, pp.754~761 (2012)
- Piersimoni C, "Current perspectives on drug susceptibility testing of Mycobacterium tuberculosis complex: the automated nonradiometric systems", J. Clin. Microbiol, 44, pp.20~28 (2006)
- Rondon L, "Evaluation of fluoromycobacteriophages for detecting drug resistance in Mycobacterium tuberculosis", J. Clin. Microbiol, 49, pp.1838~1842 (2011)
- Telenti A, "Genotypic assessment of isoniazid and rifampin resistance in Mycobacterium tuberculosis: a blind study at reference laboratory level", J. Clin. Microbiol, 35, pp.719~723 (1997)
- Toit K, "The colour test for drug susceptibility testing of Mycobacterium tuberculosis strains", Int. J. Tuberc. Lung Dis, 16, pp.1113~1118 (2012)
- Varma-Basil M, "Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay", J. Clin. Microbiol, 42, pp.5512~5516 (2004)
- Williams DL, "Contribution of rpoB mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis", Antimicrob. Agents Chemother, 42, pp.1853~1857 (1998)