Fluorescent-based PCR detection of Mycobacterium avium subsp. paratuberculosis in bovine feces and meat

A real-time fluorescent multiplex polymerase chain reaction (PCR) assay has been developed for detecting Mycobacterium avium subsp. paratuberculosis (MAP) in bovine feces and meat. This assay amplifies both an internal sequence of the IS900 gene in MAP and an internal control targeting the Ruminant specific (mt-cyt-b) gene, in order to control for any false negative results. However, PCR testing of MAP is problematic due to the presence of inhibitors and DNA extraction methods that are often laborious, hazardous, and inefficient. The Whatman FTARTM card technology was used for DNA extraction facilitating the removal of PCR inhibitors and minimizing cross contamination. This allows for high sample throughput analysis and sample storage for up to 14 years. Chelex-100 is also used to remove any remaining inhibitors and elutes MAP DNA from the FTARTM card for PCR analysis.; Detection of MAP DNA from bovine feces spiked with known concentrations of viable MAP was obtained. The detection limits of the assay was between 102 and 104 colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive results.; The sensitivity of this assay on spiked ground beef is 100 CFU/10g and the sensitivity of conventional culture is 104 CFU/10g. Furthermore, a survey on 200 retail ground beef samples using this system was performed and did not detect the presence of MAP.; Finally, three DNA extraction methods were analyzed to determine the detection limits of MAP spiked into bovine feces over a period of 30 trials. The FTA card system was able to detect 102 - 103 CFU/g MAP in all repeats with some samples containing 10 CFU/g could be detected. When using the MagNA Pure LC System alone, some samples containing 10 2 - 103 CFU/g could be detected. Utilizing a combination of the FTA card technology and the MagNA Pure LC System, the detection limit was 102 - 103 CFU/g with some samples containing 10 CFU/g yielded positive results. Among the three extraction methods analyzed, the FTA card technology coupled with the MagNA Pure LC System is the most effective way to extract MAP DNA and to remove PCR inhibitors.