Current Methods in the Molecular Typing of Mycobacterium tuberculosis and Other Mycobacteria
Figure 1
Schematic representation of the chromosome of a hypothetical Mycobacterium tuberculosis complex isolate with marked repetitive elements as targets for different typing methods. The principle of those methods is pictorially outlined. (1) In IS6110-RFLP typing, mycobacterial DNA is cleaved with the restriction endonuclease PvuII, and the resulting fragments are separated electrophoretically on an agarose gel, transferred onto a nylon membrane by Southern blotting, and hybridized to a probe complementary to the 3′-end of the IS6110 (probe target) yielding a characteristic banding pattern, in which every band represents a single IS6110 element. (2) Spoligotyping relies upon PCR amplification of a single direct repeat (DR) locus which harbours 36 bp direct repeats interspersed with unique 34–41 bp spacer sequences. The PCR products (red horizontal lines) are hybridized to a membrane containing 43 oligonucleotides corresponding to the spacers from M. tuberculosis H37Rv and Mycobacterium bovis BCG. The presence or absence of each of those 43 spacers in the DR region of the analysed isolate will be represented as the pattern of positive or negative hybridization signals. (3) The variable numbers of tandem repeat loci (VNTR) or mycobacterial interspersed repetitive units (MIRU) are PCR-amplified and the obtained products (yellow horizontal line) are sized on agarose gels to deduce the number of repeats in each individual locus. (4, 5) Two PCR-based typing methods, that is, double-repetitive-element PCR (DRE-PCR) and amplityping, are designed to amplify DNA between clusters of IS6110 and polymorphic GC-rich sequences (PGRS) or between clusters of IS6110 elements, respectively. Different distances between the repetitive elements and their different copy numbers result in variability of banding patterns, composed of DNA fragments amplified (a–d) and produced for individual isolates. (6) A heminested inverse PCR (HIP) depends on the amplification of the 5′-end of the IS6110 sequence along with its upstream flanking sequence, bordered by the closest BsrFI site. The size and number of PCR amplicons generated depend on the number of copies of IS6110. (7) Ligation-mediated PCR (LM-PCR) procedure allows, by introducing specifically designed linkers, amplifying the flanking sequences on both sides of the IS6110 element. Names and positions of the PCR primers were excerpted from the original papers. For more details, read the text.
