Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR

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Abstract
Immunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection ofMycobacterium aviumsubsp.paratuberculosisfrom milk and/or fecal samples from cattle and American bison. Samples were spiked withM. aviumsubsp.paratuberculosisorganisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detectM. aviumsubsp.paratuberculosisin feces and milk. By use of this simple and rapid technique, 10 or fewerM. aviumsubsp.paratuberculosisorganisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.