title: A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp. paratuberculosis using SYBR Green and the Light Cycler.
abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, and may also contribute to the onset and development of Crohn's disease in humans. Due to its reported heat resistance, isolation from pasteurised milk and the potential for transmission of MAP through environmental sources, rapid detection is imperative. Here, we present a rapid real time PCR assay for MAP that can simultaneously detect and quantify this organism in the laboratory using SYBR Green and the Lightcycler (LC). This method uses the highly characterised and specific P90, P91 primers, which amplify a 400-bp region of the IS900 element. Using quantified standard DNA, this assay can accurately detect as few as 20 copies (equivalent to approximately 1.5 organisms). The method is effective using a variety of templates including isolated MAP DNA, pure colonies or liquid culture sources, and can also work in the presence of contaminating bacteria. A useful application of this assay is the ability to accurately and rapidly quantitate the number of MAP cells in liquid culture as determined by comparison to previously enumerated standards.
authors.affiliation: Microbiology Department, National University of Ireland, Cork, Ireland.
authors.forename: Jim
authors.initials: J
authors.lastname: O'Mahony
authors.forename: Colin
authors.initials: C
authors.lastname: Hill
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D003959:Diamines; D005456:Fluorescent Dyes; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D009930:Organic Chemicals; D010283:Paratuberculosis / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I