Quantitative PCR for Listeria monocytogenes with colorimetric detection.
An enzyme-linked immunosorbent assay (ELISA)-mediated polymerase chain reaction (PCR) technique was developed to detect and quantify Listeria monocytogenes in food products. The bacterial DNA was extracted from artificially contaminated food and co-amplified with a synthetic internal standard (IS) using primers specific for the target gene coding for the invasive-associated protein (i.a.p.), a virulence factor of L. monocytogenes (i.a.p.) or IS in the presence of fluorescein-dUTP PCR products were hybridized with biotinylated probes designed for the i.a.p. or IS, and then the hybrids were bound to a streptavidin-coated ELISA plate. An alkaline phosphatase-conjugated antibody to fluorescein was added to the plate and in the presence of substrate, PCR products were quantitated based on an optical density reading. The detection limit for L. monocytogenes experimentally inoculated into milk samples and channel catfish fillets was 20 CFU/ml and 1-2 CFU/g, respectively. Little or no cross reaction was detected in the presence of other spoilage and pathogenic organisms such as Pseudomonas aeruginosa, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. The ELISA-mediated PCR technique, when compared to traditional methods, is more rapid (2 working days) for detecting and quantifying L. monocytogenes.