A Simple, Single Triplex PCR of IS6110, IS1081, and 23S Ribosomal DNA Targets, Developed for Rapid Detection and Discrimination of Mycobacterium from Clinical Samples
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/ discrimination of this bacterium from clinical samples.
Abstract
Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/ discrimination of this bacterium from clinical samples.
참고문헌 (37)
- Alcaide F, "Evaluation of the BACTEC MGIT 960 and the MB", BacT systems for recovery of mycobacteria from clinical specimens and for species identification by DNA AccuProbe, 2000, pp.38, 398~401
- Britten RJ, "The majority of human genes have regions repeated in other human genes", Proc. Natl. Acad. Sci. USA, 102, pp.5466~5470 (2005)
- Cegielski JP, "Comparison of PCR, culture, and histopathology for diagnosis of tuberculous pericarditis", J. Clin. Microbiol, 35, pp.3254~3257 (1997)
- Collins DM, "DNA fingerprinting of Mycobacterium bovis strains by restriction fragment analysis and hybridization with insertion elements IS1081 and IS6110", J. Clin. Microbiol, 31, pp.1143~1147 (1993)
- Collins DM, "Identification of an insertion sequence, IS1081, in Mycobacterium bovis", FEMS Microbiol. Lett, 67, pp.11~15 (1991)
- Drobniewski FA, "Modern laboratory diagnosis of tuberculosis", Lancet Infect. Dis, 3, pp.141~147 (2003)
- Eisenach KD, "Repetitive DNA sequences as probes for Mycobacterium tuberculosis", J. Clin. Microbiol, 26, pp.2240~2245 (1988)
- Falkinham JO 3rd, "Epidemiology of Mycobacterium avium infections in the pre- and post-HIV era", Res. Microbiol, 145, pp.169~172 (1994)
- Fomukong NG, "Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110", Tuber. Lung Dis, 75, pp.435~440 (1994)
- Kidane D, "Identification of the causative organism of tuberculous lymphadenitis in Ethiopia by PCR", J. Clin. Microbiol, 40, pp.4230~4234 (2002)
- Kim BJ, "Differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria by duplex PCR assay using the RNA polymerase gene (rpoB)", J. Clin. Microbiol, 42, pp.1308~1312 (2004)
- Kirschner P, "Species identification of mycobacteria using rDNA sequencing", Methods Mol. Biol, 101, pp.349~361 (1998)
- Klemen H, "Multiplex polymerase chain reaction for rapid detection of atypical mycobacteria and Mycobacterium tuberculosis complex", Diagn. Mol. Pathol, 7, pp.310~316 (1998)
- Kolk AH, "Detection of Mycobacterium tuberculosis in clinical samples by using polymerase chain reaction and a nonradioactive detection system", J. Clin. Microbiol, 30, pp.2567~2575 (1992)
- Kurabachew M, "Sequence analysis in the 23S rDNA region of Mycobacterium tuberculosis and related species", J. Microbiol. Methods, 54, pp.373~380 (2003)
- Lee H, "Novel polymorphic region of the rpoB gene containing Mycobacterium speciesspecific sequences and its use in identification of mycobacteria", J. Clin. Microbiol, 41, pp.2213~2218 (2003)
- McNabb A, "Direct identification of mycobacteria in primary liquid detection media by partial sequencing of the 65-kilodalton heat shock protein gene", J. Clin. Microbiol, 44, pp.60~66 (2006)
- Minh NN, "Molecular characteristics of rifampin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Vietnam", J. Clin. Microbiol, 50, pp.598~601 (2012)
- Mokaddas E, "Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from nontuberculous mycobacteria", Jpn. J. Infect. Dis, 60, pp.140~144 (2007)
- Mustafa AS, "Detection of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria by multiplex polymerase chain reactions", East Mediterr. Health J, 5, pp.61~70 (1999)
- Pietrzak J, "Comparison of polymerase chain reaction with standard methods in the diagnosis of Mycobacterium tuberculosis infection", Eur. J. Clin. Microbiol. Infect. Dis, 13, pp.1079~1083 (1994)
- Querol JM, "Rapid diagnosis of pleural tuberculosis by polymerase chain reaction", Am. J. Respir. Crit. Care Med, 152, pp.1977~1981 (1995)
- Reimer LG, "Laboratory detection of mycobacteremia", Clin. Lab. Med, 14, pp.99~105 (1994)
- Rivero A, "rate of tuberculosis reinfection during a nosocomial outbreak of multidrug-resistant tuberculosis caused by Mycobacterium bovis strain B", Clin. Infect. Dis, 32, pp.159~161 (2001)
- Roth A, "Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences", J. Clin. Microbiol, 36, pp.139~147 (1998)
- Shah DH, "A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis", FEMS Microbiol. Lett, 214, pp.39~43 (2002)
- Singh KK, "Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy", J. Clin. Pathol, 53, pp.355~361 (2000)
- Skuce RA, "Genomic fingerprinting of Mycobacterium bovis from cattle by restriction fragment length polymorphism analysis", J. Clin. Microbiol, 32, pp.2387~2392 (1994)
- Society AT, "Diagnostic standards and classification of tuberculosis and other mycobacterial diseases", Am. Rev. Respir. Dis, 123, pp.343~358 (1981)
- Thierry D, "IS6110, an IS-like element of Mycobacterium tuberculosis complex", Nucleic Acids Res, 18, pp.188~ (1990)
- van Embden JD, "Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology", J. Clin. Microbiol, 31, pp.406~409 (1993)
- van Soolingen D, "Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains:evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis", J. Clin. Microbiol, 29, pp.2578~2586 (1991)
- van Soolingen D, "Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG", J. Clin. Microbiol, 30, pp.1772~1777 (1992)
- Wang JY, "Performance assessment of a nested-PCR assay (the RAPID BAP-MTB) and the BD ProbeTec ET system for detection of Mycobacterium tuberculosis in clinical specimens", J. Clin. Microbiol, 42, pp.4599~4603 (2004)
- Global tuberculosis control, "World Health Organization", World Health Organization, ~ (2011)
- Yang ZH, "Restriction fragment length polymorphism Mycobacterium tuberculosis strains isolated from Greenland during 1992: evidence of tuberculosis transmission between Greenland and Denmark", J. Clin. Microbiol, 32, pp.3018~3025 (1994)
- Zelazny AM, "Identification of Mycobacterium species by secA1 sequences", J. Clin. Microbiol, 43, pp.1051~1058 (2005)