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United States Department of Agriculture

Agricultural Research Service

Title: Twelve Hour Real-Time Pcr Technique for the Sensitive and Specific Detection of Salmonella in Raw and Ready-to-Eat Meat Products

Authors
item Ellingson, J - MARSHFIELD CLINIC
item Anderson, J - MARSHFIELD CLINIC
item Carlson, Steven
item Sharma, Vijay

Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 20, 2003
Publication Date: February 20, 2004
Citation: ELLINGSON, J.L., ANDERSON, J.L., CARLSON, S.A., SHARMA, V.K. TWELVE HOUR REAL-TIME PCR TECHNIQUE FOR THE SENSITIVE AND SPECIFIC DETECTION OF SALMONELLA IN RAW AND READY-TO-EAT MEAT PRODUCTS. MOLECULAR AND CELLULAR PROBES. 2004. V. 18. P. 51-57.

Interpretive Summary: Rapid pathogen testing is vital to the food industry. Current methods provide reliable negative results in 48 hours, but a presumptive positive result must be confirmed by traditional culture methods, requiring an additional 72 hours. Polymerase Chain Reaction (PCR) testing technology is accepted as an accurate diagnostic tool; however, traditional PCR techniques can require several days. We sought to develop a rapid PCR technique for use in detecting Salmonella species in food products. Composite DNA was extracted and used as template for PCR amplification in the LightCycler**TM PCR instrument (Roche Diagnostics). Salmonella was detected down to 1 organism/ml in food products after incubation and concentration. The same samples were tested by traditional methods and correlated 100% to those of our PCR. PCR methods using the LightCycler**TM can detect and confirm the presence or absence of Salmonella species in raw and ready-to-eat beef products within 12 hours with increased sensitivity compared to traditional methods.

Technical Abstract: Rapid pathogen testing is vital to the food industry. Enzyme immunoassay (EIA) methods provide reliable negative results in 48 hours, but a presumptive positive (suspect) EIA result must be confirmed by traditional culture methods, requiring an additional 72 hours. Polymerase Chain Reaction (PCR) testing technology is accepted as an accurate diagnostic tool; however, traditional PCR techniques can require several days. We sought to develop a rapid, real-time quantitative PCR technique for use in detecting Salmonella species in food products. Salmonella abaetatuba was inoculated into raw and ready-to-eat beef products. Composite DNA was extracted and used as template for PCR amplification in the LightCycler**TM PCR instrument (Roche Diagnostics). Salmonella-specific PCR primers were designed to amplify a 250-bp product spanning from base 2305 to base 2555 of the Salmonella invasion protein (sip B/C) region (GenBank Accession #U25631). Fluorescence-labeled hybridization probes were designed to anneal to the upper strand from positions 2464-2497 (upstream) and 2499-2531 (downstream). Salmonella was detected down to 1 organism/ml in food products after incubation and concentration. The same samples were tested by visual immunoprecipitate (VIP) and traditional culture methods. Fifteen control strains of Salmonella were similarly analyzed, and specificity assays performed. The results of real-time PCR correlated 100% to those of VIP and culture. PCR methods using the LightCycler**TM can detect and confirm the presence or absence of Salmonella species in raw and ready-to-eat beef products within 12 hours with increased sensitivity compared to traditional culture and EIA methods.

Last Modified: 7/28/2014