Peer-Reviewed Journal Details
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O'Mahony, J,Hill, C
2002
November
Journal of Microbiological Methods
A real time PCR assay for the detection and quantitation of Mycobacterium avium subsp paratuberculosis using SYBR Green and the Light Cycler
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real time PCR detection quantitation Mycobacterium avium ssp paratuberculosis POLYMERASE-CHAIN-REACTION CROHNS-DISEASE JOHNES-DISEASE COWS MILK PASTEURIZATION TISSUES HYBRIDIZATION DIAGNOSIS CATTLE
51
283
293
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(TM) (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 similar to1.5 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. (C) 2002 Elsevier Science B.V. All rights reserved.
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