Abstract
Aims
The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture.
Methods and Results
The new method couples Map‐specific peptide‐mediated magnetic separation technique with an optimized phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method, and the dynamic range of the assay was 3 × 102–6 × 108 phage ml−1. When low numbers of Map were present (102 CFU ml−1), the burst size of a single host Map cell was maximal (103 phage per cell) resulting in a highly sensitive screening assay.
Conclusion
A rapid, sensitive immuno‐based screening method suitable for the detection of viable Map in milk and faeces was developed.
The objective of this study was to develop a novel screening method for detection of viable Mycobacterium avium subsp. paratuberculosis (Map) in milk and faeces, as a rapid alternative to Map culture.
Methods and Results
The new method couples Map‐specific peptide‐mediated magnetic separation technique with an optimized phage amplification assay followed by detection of released progeny phage by ELISA in a competition assay format using polyclonal antibody produced against the D29 mycobacteriophage involved in the phage assay. Sample matrices were found not to interfere with the developed method, and the dynamic range of the assay was 3 × 102–6 × 108 phage ml−1. When low numbers of Map were present (102 CFU ml−1), the burst size of a single host Map cell was maximal (103 phage per cell) resulting in a highly sensitive screening assay.
Conclusion
A rapid, sensitive immuno‐based screening method suitable for the detection of viable Map in milk and faeces was developed.
| Original language | English |
|---|---|
| Pages (from-to) | 808-817 |
| Journal | Journal of Applied Microbiology |
| Volume | 115 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 1 Sept 2013 |
| Externally published | Yes |