Discriminatory ability of hypervariable variable number tandem repeat loci in population-based analysis of Mycobacterium tuberculosis strains, London, UK

Abstract

To address conflicting results about the stability of variable number tandem repeat (VNTR) loci and their value in prospective molecular epidemiology of Mycobacterium tuberculosis, we conducted a large prospective population-based analysis of all M. tuberculosis strains in a metropolitan setting. Optimal and reproducible conditions for reliable PCR and fragment analysis, comprising enzymes, denaturing conditions, and capillary temperature, were identified for a panel of hypervariable loci, including 3232, 2163a, 1982, and 4052. A total of 2,261 individual M. tuberculosis isolates and 265 sets of serial isolates were analyzed by using a standardized 15-loci VNTR panel, then an optimized hypervariable loci panel. The discriminative ability of loci varied substantially; locus VNTR 3232 varied the most, with 19 allelic variants and Hunter-Gaston index value of 0.909. Hypervariable loci should be included in standardized panels because they can provide consistent comparable results at multiple settings, provided the proposed conditions are adhered to.

Figure 1

Effect of various enzymes and separation conditions on amplification and detectable molecular weights of PCR fragments for 4 variable number tandem repeat (VNTR) loci. A) Mycobacterial interspersed repetitive unit locus 26; B) locus exact tandem repeat; C) locus 1982; D) locus 3232.

Figure 2

Agarose gel showing the stability of amplified fragments of variable number tandem repeat (VNTR) 3336 from 2 serial isolates isolated from 4 patients. Lane 1, patient A, isolate 1, isolated 2005 Jun 20, 8 copies; lane 2, patient A, isolate 2, isolated 2005 Jul 11, 8 copies; lane 3, patient B, isolate 1, isolated 2005 Jul 8, 9 copies; lane 4, patient B, isolate 2, isolated 2005 Aug 8, 9 copies; lane 5, patient C, isolate 1, isolated 2005 Nov 11, 7 copies; lane 6, patient C, isolate 2, isolated 2005 Nov 15, 7 copies; lane 7, patient D, isolate 1, isolated 2005 May 16, 6 copies; lane 8, patient D, isolate 2, isolated 2005 May 25, 6 copies.