Development of Multitarget Real-Time PCR for the Rapid, Specific, and Sensitive Detection of Yersinia pestis in Milk and Ground Beef

Abstract

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.

Rapid Detection and Identification ofYersinia pestisfrom Food Using Immunomagnetic Separation and Pyrosequencing

“…These markers, if carefully selected could be used to discriminate between closely related pathogenic strains and could be used in the specific detection and identification of microbial contamination in food. The detection of Y. pestis in food matrices using real-time PCR has been previously reported [22], however, the limits of detection reported required further improvement. The potential for contamination with very low bacterial numbers in food matrices necessitates the development of methods for efficient capture from food matrices.…”

“…The use of IMS for the efficient capture of pathogens from food has received wide attention [22–26]. There is limited information on capture and detection of Y. pestis in food [22] and, therefore, a need to explore this further.…”

“…The preparation of DNA from samples captured from the different foods was done as previously described [22]. Briefly, 50  μ L of bead samples captured from food were lysed by vortexing vigorously and heating at 95°C in a thermal cycler.…”

“…Here, we present the application of IMS and pyrosequencing based assays for the rapid, specific, and sensitive detection and identification of Y. pestis from food matrices such as milk, bagged salad, and processed meat. This assay for Y. pestis detection is a significant improvement over our previous work using the Pathatrix sample preparation system and real-time PCR [22] and demonstrates better limits of detection without an enrichment step. The combination of efficient immunomagnetic concentration of biothreat agents and pyrosequence-based detection system is novel and represents the first report for detection and identification of Y. pestis in food with potential biodefence application.…”

Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection

“…Multiplexing is achieved employing different TaqMan probes labeled with spectrally distinct fluorophores. Selected recent examples [23][24][25][26][27], as well as commercially available PCR-based kits are reported in Table 1.…”