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 ؋ 10 9 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.Over a decade, the fingerprinting method based on restriction fragment length polymorphism (RFLP) of IS6110 insertion sequences has been established as the standard for typing strains of Mycobacterium tuberculosis. IS6110 RFLP fingerprinting is very powerful when it is used to classify M. tuberculosis isolates harboring a large number of IS6110 in their chromosomes (33). However, the prevalence of M. tuberculosis strains harboring no, single, or few copies of IS6110 in their chromosomes dramatically lowers the discriminating efficiency of the method (1). In this regard, many alternative RFLPbased fingerprinting methods, e.g., direct repeat (DR) and polymorphic GC-rich repetitive sequence RFLP fingerprinting, are supplementarily used for differentiation (10, 24). In addition, various methods based on PCR, for example, ligation-mediated PCR, mixed-linker PCR, double repetitive element PCR, and DR-based spoligotyping, were developed mainly in order to avoid the technical demand of RFLP (5, 11, 14, 21). However, most PCR-based methods displayed poor discrimination power compared to the standard IS6110 RFLP typing, whereas others were critically confronted with limitations with respect to reproducibility and reliability (16).Variable-number tandem repeats (VNTR), often referred to as micro-or minisatellite DNA, are ubiquitous in eukaryotes and humans. They have been extensiv...

No abstract

Cynomolgus monkeys (Macaca fascicularis; MF) are commonly used as nonhuman primate models for pharmaceutical product testing. In their habitat range, monkeys have close contact with humans, allowing the possibility of bidirectional transmission of tuberculosis (TB) between the two species. Although the intradermal tuberculin skin test (TST) is used for TB detection in MF, it has limitations. Herein, we established the mIGRA, combining human QuantiFERON-TB Gold-Plus and monkey IFN-γ ELISApro systems, and used it to investigate 39 captive MF who were cage-mates or lived in cages located near a monkey who died from the naturally TB infection. During a 12-month period of study, 14 (36%), 10 (26%), and 8 (21%) monkeys showed TB-positive results using the mIGRA, the TST, and TB culture, respectively. Among the 14 mIGRA-positive monkeys, 8 (57.1%) were TST-positive and 7 (50%) were culture-positive, indicating early TB detection in the latent and active TB stages with the mIGRA. Interestingly, 3 (37.5%) of the TST-negative monkeys were culture-positive. Our study showed that the mIGRA offers many advantages, including high sensitivity and high throughput, and it requires only one on-site visit to the animals. The assay may be used as a supplementary tool for TB screening in MF.