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Journal of Clinical Microbiology, October 2005, p. 5034-5043, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5034-5043.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Polymorphism of Variable-Number Tandem Repeats at Multiple Loci in Mycobacterium tuberculosis{dagger}

Nat Smittipat,1 Pamaree Billamas,1 Manee Palittapongarnpim,2 Arunee Thong-On,2 Mansuet M. Temu,2,{ddagger} Prateep Thanakijcharoen,3 Opart Karnkawinpong,3 and Prasit Palittapongarnpim1,2*

National Center for Genetic Engineering and Biotechnology, Phatumthani,1 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok,2 Amnat Charoen Hospital, Amnat Charoen, Thailand3

Received 22 March 2005/ Returned for modification 13 May 2005/ Accepted 24 July 2005

Genotyping based on variable-number tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of Mycobacterium tuberculosis. Here we investigate the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci, including nine novel ones, were polymorphic. Ten VNTR loci had high allelic diversity (Nei's diversity indices ≥ 0.6) and subsequently were used as the representative VNTR typing set for comparison to IS6110-based restriction fragment length polymorphism (RFLP) typing. The 10-locus VNTR set, potentially providing >2 x 109 allele combinations, obviously showed discriminating capacity over the IS6110 RFLP method for M. tuberculosis isolates with fewer than six IS6110-hybridized bands, whereas it had a slightly better resolution than IS6110 RFLP for the isolates having more than five IS6110-hybridized bands. Allelic diversity of many VNTR loci varied in each IS6110 RFLP type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may have evolved from two different lineages (high and low IS6110 copy number). In addition, we found that the lengths of many VNTR loci had statistically significant relationships to each other. These relationships could cause a restriction of the VNTR typing discriminating capability to some extent. Our results suggest that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis. The discriminating power of the VNTR typing system can still be enhanced by the supplementation of more VNTR loci.

* Corresponding author. Mailing address: Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Phayathai, Bangkok, 10400, Thailand. Phone: 66-2-5646700, ext. 3444. Fax: 66-2-6445411. E-mail: prasit{at}biotec.or.th.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{ddagger} Present address: National Institute for Medical Research, Mwanza Medical Research Center, Mwanza, Tanzania.

Journal of Clinical Microbiology, October 2005, p. 5034-5043, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5034-5043.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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