Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR

Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells, A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed, The expression of three L. monocytogenes genes, lap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR, Total RNA from L. monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes, Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes, The levels of expression of these three genes differed markedly, and the results indicated that the lap gene would provide a good target for development of a specific method for detection of viable L. monocytogenes based on RT-PCR amplification, After a l-h enrichment, the 371-bp lap-specific product was detected with a sensitivity of ca, 10 to 15 CFU/ml from pure culture, Detection of the 713-bp hly-specific amplicon was ca, 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period, The amplification of the lap mRNA was specific for L. monocytogenes. Overall, the assay could be completed in ca, 54 h, The use of RT-PCR amplification for the detection of viable L. monocytogenes was validated in artificially contaminated cooked ground beef, Following a 2-h enrichment incubation, the lap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca, 3 CFU/g, These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L. monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.