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AIM To characterize genomic DNA flanking IS900 insertions and develop a rapid Multiplex PCR IS900Locus (MPIL) typing method for MAP reporting the presence or absence of the element at each locus,METHODS Genomic DNA flanking 14 of the 18 IS900 loci was sequenced and compared with databasehomologues. An MPIL typing method was developed using a common IS900 primer and individual locus-specific primers designed to produce amplification products differing by about 50bp which could be easilyresolved on a single gel. MPIL was applied to a panel of 81 MAP isolates and compared with RFLP profiles.RESULTS Genes flanking IS900 loci included homologues of transcription regulators, a sigma factor, anitrate reductase, a polyketide synthase and an O6-methylguanine-methyl transferase. MPIL rapidly andconsistently identified 10 individual types of MAP from the panel of 81 isolates, and distinguished betweenbovine and ovine strains. Nine MPIL types corresponded directly to single RFLP types previously identified.CONCLUSION Isg00 insertions in MAP may affect the expression of genes critically associated with thepathogenic phenotype. MPIL typing can identify bovine and ovine strains independent of the need for cultureand may contribute to studies of the molecular epidemiology of these difficult organisms.
