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

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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 101 to 103 CFU/ml in milk and 102 to 105 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.

Document Type: Research Article

Affiliations: 1: Canadian Food Inspection Agency, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4 2: Canadian Food Inspection Agency, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4; Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta, Canada T1K 3M4 3: Canadian Food Inspection Agency, Lethbridge Laboratory, P.O. Box 640, Township Road 9-1, Lethbridge, Alberta, Canada T1J 3Z4; Department of Animal Science, McGill University, Macdonald-Stewart Building, 21111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9

Publication date: January 1, 2010

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    First published in 1937, the Journal of Food Protection®, is a refereed monthly publication. Each issue contains scientific research and authoritative review articles reporting on a variety of topics in food science pertaining to food safety and quality. The Journal is internationally recognized as the leading publication in the field of food microbiology with a readership exceeding 11,000 scientists from 70 countries. The Journal of Food Protection® is indexed in Index Medicus, Current Contents, BIOSIS, PubMed, Medline, and many others.

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