Detection, quantification and vitality of Listeria monocytogenes in food as determined by quantitative PCR

In this paper we describe the development of a quantitative PCR (qPCR) technique to detect, quantify and determine the vitality of Listeria monocytogenes in foods. The method was based on the amplification of the intergenic region spacer (IGS) between the 16S and 23S rRNA genes. A panel of more than 100 strains of Listeria spp. and non-Listeria was used in order to verify the specificity of the primers and Taqman probe and amplification signals were obtained only when L. monocytogenes DNA and RNA were loaded in the qPCR mix. Standard curves were constructed in several food matrices (milk, meat, soft cheese, fermented sausage, cured ham and ready-to-eat salad). The quantification limit was of 10(3)-10(4) cfu/g or ml, while for the determination of vitality it was 10(4)-10(5) cfu/g or ml. After an overnight enrichment in BHI at 37 degrees C also 10 cfu/g or ml could be detected in all the matrices used in this study. When we applied the protocol to food samples collected from the market or from small food processing plants, on a total number of 66 samples, 4 fresh cheeses from raw milk gave positive results prior to the overnight incubation, while 9 samples, of which only one represented by fresh meat and the others by cheeses from raw milk, were positive after the enrichment. Out of the 4 positive samples, only one could be quantified and it was determined to contain 4x10(3) cfu/g.