Development of multitarget real-time PCR for the rapid, specific, and sensitive detection of Yersinia pestis in milk...

Real-time PCR has been used previously to detect Yersinia pestis; this study applies this rapid, specific, and sensitive nucleic acid-based method to the detection and quantitation of Y. pestis specifically in food. Five sets of primers and corresponding TaqMan dual-labelled fluorogenic hybridization probes for Y. pestis were designed and optimized for specificity testing using genomic DNA from 71 bacterial strains. Four Y. pestis -specific primer and probe sets were developed, based on the virulence plasmid targets, and used to distinguish this bacterium from the various Yersinia and other bacterial species tested. An additional primer and probe set, based on a chromosomal gene target, distinguished Y. pestis and Yersinia pseudotuberculosis from the various Yersinia and other bacterial species tested. With optimized conditions, the quantitative detection limit of the probes for Y. pestis pure cultures ranged from 13 to 220 CFU. Standard curves were generated for the probes and used to determine the amplification efficiencies. The primers and probes demonstrated high amplification efficiencies, and their performance was evaluated using spiked milk and ground beef samples. The quantitative detection limit was 10(1) to 10(3) CFU/ml in milk and 10(2) to 10(5) CFU/g in ground beef without any preenrichment step. Testing the hybridization probes on food samples demonstrated the detection of Y. pestis in a foodborne application; this is the first such report, to our knowledge.