Genome-sequence-based fluorescent amplified-fragment length polymorphism analysis of Mycobacterium tuberculosis

The whole-genome fingerprinting technique, fluorescent amplified-fragment l ength polymorphism (FAFLP) analysis, was applied to Mycobacterium tuberculo sis. Sixty-five clinical isolates were analyzed to determine the value of F AFLP as a stand-alone genotyping technique and to compare it,vith the well- established IS6110 typing system. The genome sequence of M. tuberculosis st rain H37Rv (S, T, Cole et al,, Nature 393:537-544, 1998) was used to model computer-generated informative primer combination(s), and the precision and reproducibility of FAFLP were evaluated by comparing the results of in vit ro and computer-generated experiments. Multiplex FAFLP was used to increase resolving power in a predictable and systematic fashion. FAFLP analysis wa s broadly congruent with IS6110 typing for those strains with multiple IS61 10 copies. It was also able to resolve an epidemiologically unlinked group of strains with only one copy of IS6110; up to 10% of clinical isolates may fall into this category. For certain epidemiological investigations, it wa s concluded that a combination of FAFLP and IS6110 typing would give higher resolution than would either alone, FAFLP data were digital, precise, repr oducible, and suitable for rapid electronic dissemination, manipulation, in terlaboratory comparison, and storage in national or international epidemio logical databases. Because FAFLP samples and analyzes base substitution acr oss the genome as a whole, FAFLP could generate new information about the m icroevolution of the M, tuberculosis complex.