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.
abstract: Mycobacterium avium subsp. paratuberculosis 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 IS900 is used to detect M. avium subsp. paratuberculosis DNA. However, closely related IS900 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 M. avium subsp. paratuberculosis within 6 h by using real-time PCR. Specificity of the target was established using 40 M. avium subsp. paratuberculosis isolates, 67 different bacterial species, and two intestinal parasites. Using the probes and methods described, we detected 27 (2.09%) M. avium subsp. paratuberculosis-positive stool specimens from 1,293 individual stool samples by the use of either IS900 or probes deriving from the MAP2765c and MAP0865 genes described here. In general, bacterial load due to M. avium subsp. paratuberculosis was uniformly low in these samples and we estimated 500 to 5,000 M. avium subsp. paratuberculosis bacteria per gram of stool in assay-positive samples. Thus, the methods described here are useful for rapid and specific detection of M. avium subsp. paratuberculosis in clinical samples.
authors.affiliation: Institute of Medical Microbiology, Frankfurter Strasse 107, D-35392 Giessen, Germany.
authors.forename: Can
authors.initials: C
authors.lastname: Imirzalioglu
authors.forename: Heinrich
authors.initials: H
authors.lastname: Dahmen
authors.forename: Torsten
authors.initials: T
authors.lastname: Hain
authors.forename: Andre
authors.initials: A
authors.lastname: Billion
authors.forename: Carsten
authors.initials: C
authors.lastname: Kuenne
authors.forename: Trinad
authors.initials: T
authors.lastname: Chakraborty
authors.forename: Eugen
authors.initials: E
authors.lastname: Domann
mesh_terms: D000818:Animals; D058491:Bacterial Load; D001431:Bacteriological Techniques / Q000379:methods*; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology*; D005798:Genes, Bacterial; D006801:Humans; D007422:Intestines / Q000382:microbiology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000302:isolation & purification*; D010283:Paratuberculosis / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers