Direct, quantitative detection of Listeria monocytogenes in fresh raw whole milk by qPCR

A method previously developed for direct (non-enrichment) detection of Escherichia coli O157:H7 was adapted for Listeria monocytogenes. The sample treatment protocol was successful in concentrating bacteria from 10 mL raw milk samples and reducing PCR inhibition, but qPCR detection sensitivity and reproducibility was poor. Two DNA extraction reagents and multiple extraction conditions were tested to identify an efficient and reproducible DNA extraction method. Two primer/probe sets were evaluated at two concentrations and three annealing temperatures to minimize false-positive results and optimize sensitivity and reproducibility of qPCR detection. Under the selected conditions, DNA was extracted efficiently from the entire milk sample in a volume of 10 μL, and subsequently quantitated by a 5′ nuclease qPCR assay lasting 50 min. The method provided detection of 1 cfu mL−1L. monocytogenes in 10 mL raw milk and quantitation from 10 to 1000 cfu mL−1 with a total time to result of <3 h.