Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units

Abstract

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.

FIG. 1

Multiplex PCR analysis of the 12 MIRU-VNTR loci (top) of M. tuberculosis H37Rv with a detailed view of locus 23 (bottom). Four multiplex PCRs were performed on genomic DNA from M. tuberculosis H37Rv, and the products were loaded onto four lanes of an automated sequencer. The GeneScan software was used for the collection of the raw sizing data. The results from the analysis of these data with the Genotyper software are shown. Boxed numbers below the signal peaks, calculated sizes (in base pairs) of the labeled PCR products and the corresponding identified MIRU-VNTR alleles. Positions of stutter peaks, easily diagnosed as low-intensity PCR artifacts, are shown.

FIG. 2

Dendrogram of genetic relationships among 90 isolates of the M. tuberculosis complex from 38 countries based on the 12 MIRU-VNTR loci. A majority rule consensus tree was built using the neighbor-joining distance algorithm as described in Materials and Methods. The linkage distance scale is indicated at the bottom. M. bovis isolate 130 is discriminated from M. bovis isolate 71 based on the presence of an additional 53-bp invariable MIRU unit in locus 4 in the first strain (this unit is not taken into account in the MIRU-VNTR dendrogram analysis). Dotted lines, groups including mainly Haarlem, Africa, and Beijing isolates identified by Kremer et al. (12). The numbers of MIRU-VNTRs in locus 24 (24:n) are indicated at the right. Isolates are M. tuberculosis, except for those indicated as M bov, M afr, M can, and M mic, corresponding to M. bovis, Mycobacterium africanum, M. canettii, and M. microti isolates, respectively.