Application of a rapid DNA extraction method with real-time fluorescence PCR for the detection of Mycobactehum avium subsp. paratuberculosis in raw milk

摘要

The screening of milk for Mycobactehum avium subsp. paratuberculosis (MAP) DNA using PCR can be problematic due to the presence of agents in raw milk that can inhibit or hinder the PCR reaction. The purpose of this study was to evaluate a DNA extraction method commonly used in forensic science, for its potential application in screening for MAP DNA in bovine raw milk. Bulk tank milk from a herd repeatedly tested negative for Johne's disease was spiked with different log concentrations of MAP (from approximately 10~4 to 1 CFU/ml). Forty five milliliters of the sample was centrifuged and the pellet was placed onto FTA card media (Whatman~R) for DNA extraction. Total DNA was eluted from the card after appropriate washing steps, and real time PCR using fluorescence resonance energy transfer (FRET) probes (Roche Lightcycler~R) targeting the IS900 sequence was performed. The assay was able to reliably detect samples containing approximately 2 logs CFU/ml MAP and was able to detect samples containing only one log CFU/ml in a majority of spiked samples. The DNA extraction and PCR protocol was able to be completed in 4 hours and also lends itself well to high sample throughput and the archiving of samples. The novel protocol can potentially contribute to the improvement of MAP control programs and to the assessment of the quality and safety standards of milk and dairy products since it provides a rapid screening method for both hospital and bulk tank milk, reducing the numbers of samples to be cultured.