Objective: To evaluate the discriminatory efficiency of multiple loci of variable numbers of tandem repeats (vNTR) in Mycobacterium tuberculosis genome. Genotyping and identification on Chinese M. tuberculosis clinical strains were used to locate a series of high discriminated loci, so as to provide the basis for creating a standardized multiple loci VNTR analysis (MLVA) to distribute fast typing and identification on Chinese M. tuberculosis.
Methods: VNTR loci which were chosen from the website http//minisatellites.u-psud.fr/ and referenced to the genome sequence of M. tuberculosis standard strain H37Rv were tested in Chinese M. tuberculosis clinical strains and H37Rv by means of PCR. The primers were designed by DNAStar software. The repeat member of VNTR unit was estimated by the result of PCR. The discrimination power of single locus or multiple loci was confirmed by the Hunter-Gaston index.
Results: 45 VNTR loci were tested in 135 Chinese M. tuberculosis clinical strains and H37Rv. The discrimination power of these loci appeared different from each other, with the biggest Hunter-Gaston index as 0.814 (0.797-0.830), the smallest one as 0.015 (0.001-0.028), and there were 13 loci with which the Hunter-Gaston index was bigger than 0.5. Results showed that the discrimination power was increasing by different loci that associated with each other. The more loci that were combined, the bigger the Hunter-Gaston index was. For example, the Hunter-Gaston index of Qub11-b associated with Qub18 was 0.936, by which 136 strain could be divided into 44 groups. With the combination of 9 loci including Qub11-b, Qub18, Mtub21, Rv2372, MIRU26, Qub26, Qub4156c, Qub11-a and Qub15, the Hunter-Gaston index could have reached 1 and by which the 136 swains could be divided into 136 groups, also showing that the biggest discrimination power to strain identification, viz, strain level genotype were reached.
Conclusion: The discrimination power of different locus was different. The discrimination power of multiple loci was much more satisfied than that of single locus. It was satisfied the combine discrimination power of 9 loci including Qub11-b, Qub18, Mtub21, Rv2372, MIRU26, Qub26, Qub4156c, Qub11-a and Qub15, by which the qualified typing method could gain to facilitate research on molecular epidemiology with the Hunter-Gaston index analysis.