COMPARISON OF 3 RESTRICTION ENDONUCLEASES IN IS1245-BASED RFLP TYPINGOF MYCOBACTERIUM-AVIUM

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates, As there is no standardised method with respect to the opti mal restriction enzyme, three restriction endonucleases were tested fo r analysis of 17 human isolates, The restriction endonucleases, select ed on the basis of the physical maps of IS1245 and of the highly homol ogous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1 311, and NruI, that cleaves both IS1245 and IS1311, All the restrictio n endonucleases yielded polymorphic and complex RFLP patterns, However , BsaAI- and NruI-generated bands were more evenly distributed and eas ier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with Bsa AI or NruI yielded probe-specific restriction fragments of molecular s ize lower than expected, Moreover, digestion with NruI, which was expe cted to generate the highest numbers of bands in all the isolates, yie lded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 is olates, respectively, These findings might suggest the existence of un identified IS1245-related insertion element(s) in M. avium isolates, C omputer analysis of the IS1245-based RFLP patterns of M. avium isolate s showed that the restriction endonucleases were capable, although wit h minor differences, of defining distinct banding patterns and cluster s of identical or highly related isolates, thus confirming IS1245-base d RFLP analysis as a useful technique for epidemiological studies.