Application of 5 '-nuclease PCR for quantitative detection of Listeria monocytogenes in pure cultures, water, skim milk, and unpasteurized whole milk

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been d escribed, including quantitative PCR and the latest innovation, real-time P CR, In real-time PCR, e.g., the 5'-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Ho lland et al,, Proc. Natl. Acad. Sci, USA 88:7276-7280, 1991), We present an assay for the quantitative detection of Listeria monocytogenes based on th e 5'-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hl yA) as the target. The assay was positive for all isolates of L. monocytoge nes tested (65 isolates including the type strain) and negative for ail oth er Listeria strains (16 isolates from five species tested) and several othe r bacteria (18 species tested). The application of 5'-nuclease PCR in diagn ostics requires a quantitative sample preparation step. Several magnetic be ad-based strategies were evaluated, since these systems are simple and rela tively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approxim ately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h, In conclusion, a complete quanti tative method for L. monocytogenes in water and in skimmed and raw milk was developed.