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United States Department of Agriculture

Agricultural Research Service

Title: Using Portable Real-Time Polymerase Chain Reaction (Rt-Pcr) Assays to Detect Salmonella in Raw Milk

Authors
item Van Kessel, Jo Ann
item Karns, Jeffrey
item Perdue, Michael

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 2, 2003
Publication Date: October 1, 2003
Citation: VAN KESSEL, J.S., KARNS, J.S., PERDUE, M.L. USING PORTABLE REAL-TIME POLYMERASE CHAIN REACTION (RT-PCR) ASSAYS TO DETECT SALMONELLA IN RAW MILK. JOURNAL OF FOOD PROTECTION. 2003. pp.1762-1767.

Interpretive Summary: Traditional culture methods for detection of pathogens in foods are laboratory based, and generally time-consuming and labor intensive. Recent advancements in PCR technology may allow for more rapid, and perhaps even on-farm detection, of food-borne pathogens. The purpose of this study was to determine the efficacy of a portable RT-PCR system for the detection of Salmonella spp. in raw milk. The bulk tank milk samples (200) used in this study were a subset of a larger study and were chosen so that there were known culture-positive (24) and culture-negative (176) samples. Most of the samples (19) required enrichment in a selective medium in order to detect the presence of presumptive Salmonella but Salmonella was isolated directly from five samples. Further characterization of the presumptive Salmonella via serotyping confirmed that 22 samples were Salmonella. DNA extracts of bacterial pellets from the enriched milk samples were analyzed for Salmonella by real time polymerase chain reaction (RT-PCR) using the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.). This is a portable, self-contained instrument designed for in-field analysis. Two samples that were Salmonella-positive via traditional culture methods were Salmonella-negative based on RT-PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates were Salmonella-positive via RT-PCR. There were 32 samples that were Salmonella-negative via culture and positive via RT-PCR. Based on these results, RT-PCR may be more sensitive than traditional culture

Technical Abstract: The purpose of this study was to determine the efficacy of a portable RT-PCR system for detecting Salmonella spp. in raw milk. The bulk milk samples (200) chosen for this study were a subset of a larger study; the subset contained 24 samples that were culture-positive for Salmonella and 176 that were culture-negative. Milk was both plated directly on selective agar and enriched in selective media followed by plating. Presumptive Salmonella were isolated from direct culture of five samples while Salmonella was isolated from the remaining 19 positive samples only after enrichment. Serotyping was performed on presumptive Salmonella and isolates from 22 samples were confirmed as Salmonella. PCR assays on culture-positive milk prior to enrichment yielded no evidence of Salmonella. DNA extracts of bacterial pellets from the enriched samples were analyzed for Salmonella by RT-PCR using the Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.). Fifty-three samples were identified as Salmonella-positive from the enrichment pellets based on RT-PCR analysis. Two samples that were Salmonella-positive via culture and serotyping were Salmonella-negative based on RT-PCR. Serotyping identified isolates from these samples as Salmonella Montevideo. All DNA extracts of Salmonella Montevideo isolates were Salmonella-positive via RT-PCR. There were 32 samples that were Salmonella-negative via culture and positive via RT-PCR. Based on these results, RT-PCR may be more sensitive than traditional culture methods for the detection of Salmonella spp. in raw milk. In addition, enrichment followed by RT-PCR yields results in 24 h as opposed to 48 to 72 h for traditional culture.

Last Modified: 12/21/2014
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