High-resolution minisatellite-based typing as a portable approach to global analysis ofMycobacterium tuberculosismolecular epidemiology

Abstract

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

Use of Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing To Examine Genetic Diversity ofMycobacterium tuberculosisin Singapore

“…This method makes use of the length variation at 12 independent minisatellite-like loci scattered throughout the M. tuberculosis genome. A number of studies have proven that MIRU-VNTR typing is a reliable and reproducible typing method that enables a level of discrimination between strains that is comparable to that of IS6110 typing (7,18,24,26). The ease with which this PCR-based method can be performed, its adaptability to high-throughput automation (26), and the digital format of MIRU-VNTR patterns make this method suitable for the global study of the molecular epidemiology of M. tuberculosis.…”

“…A technique for strain typing of M. tuberculosis based on variable-number tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRU) has recently been introduced (18,25). This method makes use of the length variation at 12 independent minisatellite-like loci scattered throughout the M. tuberculosis genome.…”

“…In particular, this method is not informative in areas where the W/Beijing genotype family is prevalent, as most strains in this family share the same typical spoligotypes (3, 31). Therefore, this method is of limited use as a first-line typing method (7,15).A technique for strain typing of M. tuberculosis based on variable-number tandem repeats (VNTRs) of mycobacterial interspersed repetitive units (MIRU) has recently been introduced (18,25). This method makes use of the length variation at 12 independent minisatellite-like loci scattered throughout the M. tuberculosis genome.…”

Identifying Mycobacterium tuberculosis complex strain families using spoligotypes

“…Results of initial analysis performed on the patient demographic information according to the MTC strain families illustrated the potential for combining strain genotyping and demographic data. Future work will concentrate on developing methods for merging probabilistic models for spoligotyping and other MTC genotyping methods, such as mycobacterial interspersed repetitive units (Mazars et al, 2001;Supply et al, 1997;Supply et al, 2001) and IS6110-based restriction fragment length polymorphism analyses, with traditional epidemiological data.…”

Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC)

“…This provides important information for molecular epidemiologic analysis and assists in providing an understanding of the genetics and pathogenesis of Mycobacteria. A strain typing technique using repetitive DNA sequence interspersed in the genome, and being highly discriminatory, highly reproducible and convenient has been exploited for the M. tuberculosis complex [58,104,148,157,161] and recently adapted to M. bovis and Map [2,147].…”