Identification and sub-typing of Mycobacterium avium subsp paratuberculosis and Mycobacterium avium subsp avium by randomly amplified polymorphic DNA

A commercially available kit consisting of twenty 10-mer random primers was evaluated to allow selection of a suitable primer that would permit identi fication and sub-typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium by randomly amplified polymorphic DNA (RAP D). A primer OPE-20 (5 ' -AAC-GGT-GAC-C-3 ') was identified to be the most suitable primer when tested with four ATCC reference strains of M. paratube rculosis and eight well characterized field strains each of M, paratubercul osis and M. avium. Primer OPE-20 was further tested for its ability to iden tify and subtype 200 field isolates of M. paratuberculosis. The fingerprint patterns of M. paratuberculosis (n = 212) consisted of live unique common fragments (620, 450, 310, 230, 180 bp) and nine variable fragments resultin g in six distinct genotypes. The DNA fingerprints of M. avium (n = 8) consi sted of a single common fragment of 620 bp, and 15 variable fragments resul ting in six different genotypes. The cattle, human and goat isolates of M. paratuberculosis were genetically similar, but a sheep isolate had a differ ent RAPD profile as compared to RAPD profiles from other species. RAPD was observed to be a rapid, reproducible andreliable technique for identificati on and sub-typing of M. paratuberculosis. (C) 2001 Elsevier Science B.V. Al l rights reserved.