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Volume 146, Issue 9

Characterization of IS loci in subsp. and development of multiplex PCR typing

The GenBank accession numbers for the sequences reported in this paper are AJ011838, AJ250015–AJ250023 and AJ251434–AJ251437.

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Abstract

subsp. is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohn’s disease in humans. It differs from other members of the complex in having 14–18 copies of IS inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the and subsp. genomes were identified. These included regions encoding a sigma factor () at locus 3, a nitrate reductase () at locus 4, a transcription regulator () and polyketide synthase at locus 6, and a 6--methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS sited two bases closer to the initiation codon. IS insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one subsp. isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of subsp. , from one subsp. and from six other mycobacterial species. A multiplex PCR of IS loci (MPIL) typing method was developed which was able to discriminate 10 different types of subsp. from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single I/EII RFLP type, suggesting that this method may be applicable to typing of subsp. directly from a sample without the need for culture. The remaining MPIL type corresponded with seven I/EII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS loci.

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Keyword(s): IS900 , MPIL, multiplex PCR of IS900 loci , multiplex , Mycobacterium avium subsp. paratuberculosis and typing
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2000-09-01
2024-04-19
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Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typingThe GenBank accession numbers for the sequences reported in this paper are AJ011838, AJ250015–AJ250023 and AJ251434–AJ251437.
Microbiology 146, 2185 (2000); https://doi.org/10.1099/00221287-146-9-2185
/content/journal/micro/10.1099/00221287-146-9-2185
/content/journal/micro/10.1099/00221287-146-9-2185
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