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Imirzalioglu, Can; Dahmen, Heinrich; Hain, Torsten; Billion, Andre; Kuenne, Carsten; Chakraborty, Trinad; Domann, Eugen

Highly Specific and Quick Detection of Mycobacterium avium subsp. paratuberculosis in Feces and Gut Tissue of Cattle and Humans by Multiple Real-Time PCR Assays

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  • Media type: E-Article
  • Title: Highly Specific and Quick Detection of Mycobacterium avium subsp. paratuberculosis in Feces and Gut Tissue of Cattle and Humans by Multiple Real-Time PCR Assays
  • Contributor: Imirzalioglu, Can; Dahmen, Heinrich; Hain, Torsten; Billion, Andre; Kuenne, Carsten; Chakraborty, Trinad; Domann, Eugen
  • imprint: American Society for Microbiology, 2011
  • Published in: Journal of Clinical Microbiology
  • Language: English
  • DOI: 10.1128/jcm.01492-10
  • ISSN: 0095-1137; 1098-660X
  • Keywords: Microbiology (medical)
  • Origination:
  • Footnote:
  • Description: <jats:title>ABSTRACT</jats:title> <jats:p> <jats:named-content content-type="genus-species">Mycobacterium avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> is the causative agent of Johne's disease (JD) in cattle and may be associated with Crohn's disease (CD) in humans. It is the slowest growing of the cultivable mycobacteria, and culture from clinical, veterinary, food, or environmental specimens can take 4 months or even longer. Currently, the insertion element IS <jats:italic>900</jats:italic> is used to detect <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> DNA. However, closely related IS <jats:italic>900</jats:italic> elements are also present in other mycobacteria, thus limiting its specificity as a target. Here we describe the use of novel primer sets derived from the sequences of two highly specific single copy genes, MAP2765c and MAP0865, for the quantitative detection of <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> within 6 h by using real-time PCR. Specificity of the target was established using 40 <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> -positive stool specimens from 1,293 individual stool samples by the use of either IS <jats:italic>900</jats:italic> or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> was uniformly low in these samples and we estimated 500 to 5,000 <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of <jats:named-content content-type="genus-species">M. avium</jats:named-content> subsp. <jats:named-content content-type="genus-species">paratuberculosis</jats:named-content> in clinical samples. </jats:p>
  • Access State: Open Access