|GARANZONI, GIAN - University Of Bologna, Italy|
|ALBONETTI, SABRINA - University Of Bologna, Italy|
Submitted to: International Dairy Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/12/2014
Publication Date: 2/1/2015
Publication URL: http://handle.nal.usda.gov/10113/61021
Citation: Paul, M., Garanzoni, G.M., Albonetti, S., Brewster, J.D. 2015. Direct, quantitative detection of Listeria monocytogenes in fresh raw whole milk by qPCR. International Dairy Journal. 41:46-49. DOI: 10.1016/j.dairyj.2014.09.008.
Interpretive Summary: Raw milk is becoming increasingly popular among American consumers based on the idea that it may be healthier and more nutritious than pasteurized milk. Unfortunately, pathogens that are normally killed by the pasteurization process may be present in raw milk in sufficient numbers to cause disease. There are methods available that can determine the presence but not the quantity of pathogens in milk. In this study, an improved method was developed for the fast, sensitive measurement in raw milk of Listeria monocytogenes, a pathogen that can cause serious disease and fatalities in immuno-compromised patients. This information provides a better understanding of and ability to control the health risks associated with raw milk.
Technical Abstract: The development and optimization of a method to detect and quantify Listeria monocytogenes in raw milk is described here. Three-step treatment of samples with EDTA, SDS, DNase and trypsin was combined with centrifugation to concentrate bacteria from 10 mL of raw milk and reduce or eliminate potential PCR inhibitors. Two DNA extraction reagents and multiple temperature regimes were tested to identify the most efficient and reproducible DNA extraction method. Two primer/probe sets were evaluated at two concentrations and three annealing temperatures to optimize sensitivity and reproducibility of L. monocytogenes detection. Under optimized conditions, DNA from the entire milk sample was efficiently extracted in a total volume of 11 ul, and subsequently quantitated by a 40 min 5’ nuclease qPCR assay. The method provides detection of 1 cfu mL-1 L. monocytogenes in 10 mL raw milk and quantitation from 10 – 1000 cfu mL-1 with results obtained in less than 3 h.