Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells

In order to introduce specificity forMycobacterium avium subsp.paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) ofM. avium subsp.paratuberculosis cells and the percentage of nonspecific binding by otherMycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesizedM. avium subsp.paratuberculosis -specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture ofM. avium subsp.paratuberculosis and minimal (<1%) nonspecific recovery of otherMycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 103 to 104 CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detectM. avium subsp.paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD50 ) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viableM. avium subsp.paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.