Automated high-throughput mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis strains by a combination of PCR and nondenaturing high-performance liquid chromatography - PubMed (original) (raw)

Automated high-throughput mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis strains by a combination of PCR and nondenaturing high-performance liquid chromatography

Jason T Evans et al. J Clin Microbiol. 2004 Sep.

Abstract

Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex isolates is portable, 100% reproducible, and highly discriminatory. Nondenaturing high-performance liquid chromatography (non-dHPLC) with use of a WAVE microbial analysis system is a promising method of PCR amplicon analysis as it is low cost and requires no preanalysis processing. The aims of this study were to validate the application of WAVE microbial analysis system technology to MIRU-VNTR typing. A collection of 70 strains were cultivated in liquid culture and extracted using the QIAamp DNA minikit. Novel primers were designed to target the 12 MIRU-VNTR loci (P. Supply et al., J. Clin. Microbiol. 39:3563-3571, 2001). After amplification, each PCR product was analyzed on a WAVE microbial analysis system. The fragment size was calculated from the chromatogram, and the number of tandem repeats at each locus was determined. For the collection of 70 strains 100% concordance was achieved when comparing MIRU-VNTR profiles obtained from agarose gel electrophoresis and PCRs analyzed on a WAVE microbial analysis system. The calculated fragment sizes, obtained from the WAVE microbial analysis system, were sufficiently accurate to ensure 100% confidence when assigning the number of tandem repeats to a MIRU-VNTR locus. This study is the first to report the successful use of non-dHPLC for screening for variations in the number of MIRU-VNTRs in mycobacterial DNA. Non-dHPLC analysis was demonstrated to be a rapid, low-labor input method for the detection and analysis of MIRU-VNTR amplicons. The combination with non-dHPLC further enhances the utility of MIRU-VNTR typing.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

MIRU typing results obtained for each of the 70 M. tuberculosis strains analyzed. The dendrogram was constructed with use of the categorical coefficient algorithm and produced via UPGMA.

Similar articles

Cited by

References

    1. Braden, C. R., J. T. Crawford, and B. A. Schable. 2002. Quality assessment of Mycobacterium tuberculosis genotyping in a large laboratory network. Emerg. Infect. Dis. 8:1210-1215. - PMC - PubMed
    1. Butler, W. R., W. H. Haas, and J. T. Crawford. 1996. Automated DNA fingerprinting analysis of M. tuberculosis using fluorescent detection of PCR products. J. Clin. Microbiol. 34:1801-1803. - PMC - PubMed
    1. Collyns, T. A., D. M. Gascoyne-Binzi, and P. M. Hawkey. 2002. Molecular fingerprinting of Mycobacterium tuberculosis: does it help in understanding the epidemiology of tuberculosis? Rev. Med. Microbiol. 13:119-127.
    1. Cooksey, R. C., G. P. Morlock, B. P. Holloway, J. Limor, and M. Hepburn. 2002. Temperature-mediated heteroduplex analysis performed by using denaturing high-performance liquid chromatography to identify sequence polymorphisms in Mycobacterium tuberculosis complex organisms. J. Clin. Microbiol. 40:1610-1616. - PMC - PubMed
    1. Cowan, L. S., L. Mosher, L. Diem, J. P. Massey, and J. T. Crawford. 2002. Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units. J. Clin. Microbiol. 40:1592-1602. - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources