title: Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR.
abstract: 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, iap, 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 iap 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 1-h enrichment, the 371-bp iap-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 iap 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 iap-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.
authors.affiliation: Microbial Food Safety Research Unit, Eastern Regional Research Center, U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038, USA.
authors.forename: P G
authors.initials: PG
authors.lastname: Klein
authors.forename: V K
authors.initials: VK
authors.lastname: Juneja
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D008089:Listeria monocytogenes / Q000235:genetics / Q000302:isolation & purification*; D008460:Meat / Q000382:microbiology; D010454:Peptide Termination Factors; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010454:Peptide Termination Factors; D015534:Trans-Activators