Introduction.

Reports have indicated that M. avium subsp. paratuberculosis (MAP) survives high-temperature short-time pasteurization. Therefore, to ensure the safety of milk and dairy products it is important to detect viable MAP. The aim of this study is to develop a rapid molecular diagnostic assay for detecting viable MAP cells using PCR and NASBA assays for the SsrA gene and it's RNA transcript, tmRNA.

Methods.

Based on the SsrA gene, primers and DNA probes were designed for the amplification and detection of PCR and NASBA products to distinguish between the species of the M. avium complex. PCR assays were performed on DNA from mycobacterial species and non-mycobacterial species. Southern blot analysis confirmed specificity and sensitivity of DNA primers and probes. NASBA assays were performed on RNA extracted from mycobacterial species using Hybaid ribolyser method with analysis performed using NucliSens technology.

Results.

Universal PCR primers amplified ~350bp of the SsrA gene from members of the mycobacteria genus. A specific DNA probe (PAV1) was designed and developed to detect members of the M. avium complex and a further DNA probe (PAV2) was developed to specifically detect MAP and M. avium only. The SsrA sequences for M. avium and MAP were found to be 100% homolgous and therefore indistinguishable. NASBA amplification of RNA from a range of mycobacteria with primers designed from tmRNA followed by detection with probe PAV1 detected M. intracellulare, M. avium and MAP. Whereas PAV2 detected only the latter two.

Discussion.

The application of the tmRNA NASBA assay for monitoring the presence of viable MAP cells in raw and pasteurised milk samples is currently under investigation. We propose that this assay could be used initially to identify the presence of viable MAP and/or M. avium in milk samples. The application of an IS900 molecular assay could then be applied to distinguish between MAP and M. avium.