Detection and enumeration of Listeria monocytogenes in fresh cut vegetables using MPN-Real-Time PCR

Listeria monocytogenes is a gram positive, rod shaped, pathogenic bacterium, causative agent of a severe infection generally known as listeriosis. Packaging and storage conditions of fresh cut vegetables may favour the growth of this psychrotrophic pathogen leading to potential health threat. Detection and enumeration of L. monocytogenes in concentrations up to 103 CFU/g, usually implies use of the most-probable-number technique (MPN) which may take up to seven days for verified identification of the pathogen. We developed a fast and reliable protocol combining MPN with a Real-Time quantitative PCR (qPCR) approach. Samples of fresh cut salads (25 g) purchased at local shops were spiked with 1 to 105 CFU/g of L. monocytogenes. Samples were homogenized, and triplicate series of tubes containing 10-5 to 10 g of food were incubated in Fraser broth at 30\ub0C for 48 h for standard MPN analysis. After incubation, broth samples were taken from each tube and DNA was extracted. DNA from enrichment tubes was used as template in a qPCR assay targeting a 64 bp hlyA gene sequence of L. monocytogenes. Results of this assay were than compared with those of standard MPN analysis and a complete accordance was observed. Furthermore, we tested an enrichment free approach using the same qPCR assay. Samples were prepared as described for MPN-qPCR while DNA extraction was performed prior to enrichment of inoculated salads. This approach allowed us to identify L. monocytogenes in samples spiked with 10-105 CFU/g. The whole process, including DNA extraction, required less than four hours, thus providing a fast and reliable tool for detection of L. monocytogenes in fresh cut vegetables