Evaluation of Amplified Fragment Length Polymorphism Analysis for Inter- and Intraspecific Differentiation of

The usefulness of amplified fragment length polymorphism (AFLP) analysis was evaluated for the discrim-ination of Mycobacterium bovis (17 strains), M. tuberculosis (15 strains), and M. ulcerans (12 strains) at the inter- and intraspecific level. The AFLP technique is a whole-genome coverage genotypic fingerprinting method based on the selective PCR amplification of modified restriction fragments obtained through a double enzy-matic digest and subsequent ligation of double-stranded restriction site-specific adapter oligonucleotides. Selective amplification of ApaI/TaqI templates with primer combination A02-T02 (both having an additional C at their 3 * end) generated autoradiographic AFLP fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing clear separation of M. ulcerans (cluster I) from the M. tuberculosis complex members M. bovis and M. tuberculosis (cluster II). Calculation of similarities using the band-based Dice correlation coefficient instead of the Pearson product-moment correlation coefficient revealed a further sub-grouping in cluster II. The two resulting subclusters corresponded with the phenotypic identity of M. bovis and M. tuberculosis, respectively, and could also be visually identified by two AFLP marker bands. Because of the relatively low degree of genotypic variation among the AFLP band patterns of the latter two taxa, no correlation could be found with previously reported molecular typing data or with geographical origin. The use of primer combination A02-T01 (the latter having an A as selective base) did not increase the resolving power within th