Bacteriophage-based methods for the rapid detection of viable Mycobacterium avium subsp. paratuberculosis (MAP) in veterinary specimens are a recent addition to the Johne’s disease diagnostic toolbox. Here we report the use of D29 mycobacteriophage-coated tosylactivated paramagnetic beads to capture and concentrate MAP cells from samples (termed phagomagnetic separation, PhMS) and then naturally lyse viable MAP cells (from the inside out) to provide DNA for IS900 qPCR purposes. Transmission electron microscopy confirmed that D29 phages had bound to beads in the correct orientation and that the phage-coated beads captured MAP cells from a suspension. During test optimization, conventional IS900 PCR results were used to subjectively assess the effect of different phage: bead coating ratios, differing amounts of coated beads during PhMS, optimal incubation time post-PhMS to obtain maximal MAP DNA, and the potential benefit of a brief heat shock (55oC/1 min) prior to IS900 Taqman qPCR. The limit of detection 50% (LOD50%) of the optimized PhMS-qPCR assay was 10.00 MAP cells/50 ml milk (95% CI: 1.20–82.83). Bulk tank milk from 100 dairy farms was tested, 49 (49%) of which tested PhMS-qPCR positive, with viable MAP numbers detected ranging from 3-126 MAP/50 ml, and nine (9%) tested milk-ELISA positive; six milk samples tested positive by both tests. Kappa test indicated slight agreement between PhMS-qPCR and milk-ELISA results (Kappa 0.065, 95% CI: -0.050 to 0.179). The novel PhMS-qPCR assay is a sensitive, specific and easy-to-apply phage-based assay for viable MAP, with potential application for milk surveillance or diagnosis of Johne’s disease.
- Mycobacterium avium subsp. paratuberculosis
- phagomagnetic separation
- quantitative PCR
- viability assay
ASJC Scopus subject areas
- Food Animals
- Agricultural and Biological Sciences (miscellaneous)