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Journal of Clinical Microbiology, September 2004, p. 4175-4180, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4175-4180.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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,1* Peter M. Hawkey,1,2 E. Grace Smith,1 Kerstin A. Boese,1 Roderic E. Warren,3 and George Hong4

West Midlands Public Health Laboratory, Health Protection Agency, Birmingham Heartlands Hospital,1 Division of Immunity and Infection, University of Birmingham, Birmingham,2 Department of Microbiology, Royal Shrewsbury Hospital, Shrewsbury, United Kingdom,3 Transgenomic, Inc., Omaha, Nebraska4

Received 27 June 2003/ Returned for modification 9 September 2003/ Accepted 10 May 2004

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.

* Corresponding author. Mailing address: West Midlands Public Health Laboratory, Health Protection Agency, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, United Kingdom. Phone: 44 (0) 121-424-0250. Fax: 44 (0) 121-772-6229. E-mail: Jason.Evans{at}heartsol.wmids.nhs.uk.

Journal of Clinical Microbiology, September 2004, p. 4175-4180, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4175-4180.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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