USE OF REAL-TIME POLYMERASE CHAIN REACTIONS(PCR) FOR THE DETECTION OF PATHOGENIC MICROBES IN BULK TANK MILK

Authors: Karns, Jeffrey; Van Kessel, Jo Ann; Perdue, Michael

Submitted to: Joint Abstracts of the American Dairy Science and Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 6/25/2003
Publication Date: 6/25/2003
Citation: Karns, J.S., Van Kessel, J.S., Perdue, M.L. 2003. Use of Real-Time Polymerase Chain Reactions(PCR) for the detection of pathogenic microbes in bulk tank milk. Joint Abstracts of the American Dairy Science and Society of Animal Science. p.369

Interpretive Summary:

Technical Abstract: Recent reports suggest an increase in consumption of raw milk and products made from raw milk in the United States. Several outbreaks of food-borne disease have been associated with the consumption of these products. Traditional culture methods for detection of pathogens in foods are generally time-consuming and labor intensive, often requiring more than 96 hours for positive identification. Methods for the rapid detection of pathogenic microbes in raw milk could help to minimize risks associated with consumption of raw milk. The objective of this study was to examine the usefulness of real-time PCR for the detection of Salmonella enterica and Listeria monocytogenes in bulk-tank milk. Twenty-four milk samples identified as Salmonella positive by traditional culture techniques and 176 that were Salmonella negative based on culture techniques were chosen for PCR analysis. DNA was isolated from the same tetrathionate enrichments used for culture identification and subjected to real-time PCR analysis using a commercially available reagent kit. Fifty-three samples were identified as Salmonella-positive by real-time PCR analysis. Two samples that were identified as being positive for Salmonella via culture were identified as being Salmonella-negative based on real-time PCR while 23 samples originally determined to be Salmonella-negative were Salmonella-positive based on real-time PCR. Serotyping confirmed that isolates from the 2 cultures that were PCR negative were not Salmonella while more rigorous culture of one of the PCR-positive, culture-negative enrichments did result in isolation of Salmonella. Eighty-one samples of bulk-tank milk shown to be positive for Listeria sp. by traditional culture were chosen for analysis with a published TaqMan primer/probe set specific for Listeria monocytogenes. DNA was isolated from the same Modified Listeria Enrichment broth cultures used for culture identification. Of these 81 samples 42 were clearly positive by real-time PCR, 8 were tentatively positive, and 31 were shown not to contain L. monocytogenes, indicating that the Listeria isolated from them were non-pathogenic species. This study suggests that real-time PCR techniques can be used to detect pathogenic microorganisms in bulk-tank milk with a sensitivity as good or better than traditional culture methods. In addition, these methods yield results within 24 h for Salmonella and 48 h for Listeria, greatly reducing the time required for positive identification of these pathogens in raw milk.