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Applied and Environmental Microbiology, August 2004, p. 4561-4568, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4561-4568.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

Jim O'Mahony^* and Colin Hill

Microbiology Department and Alimentary Pharmabiotic Centre, National University of Ireland, Cork, Ireland

Received 10 December 2003/ Accepted 16 April 2004

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg²⁺ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 10⁶ to 3 x 10¹ copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.

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* Corresponding author. Mailing address: Microbiology Department and Alimentary Pharmabiotic Centre, National University of Ireland, Cork, Ireland. Phone: 353 21 490137. Fax: 353 21 4903101. E-mail: j.omahony{at}ucc.ie.

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Applied and Environmental Microbiology, August 2004, p. 4561-4568, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4561-4568.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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