Evaluation of Reverse Transcriptase 5' Nuclease Polymerase Chain Reaction assay for the Detection of Viable Heat-Injured and Resuscitated Listeria monocytogenes in Ground Pork

An anaerobic resuscitation-enrichment system was combined with a 5' nuclease reverse transcriptase (RT) protocol for detecting Listeria monocytogenes Scott A from artificially inoculated ground pork. When irradiation-sterilized ground pork containing L. monocytogenes (~6 x 10 5 CFU/g) was heated (60 o C, 14 min), 100% of the cells were injured, as indicated by no growth on selective Modified Oxford (MOX) agar plates incubated aerobically. After resuscitation and enrichment (37°C) in anaerobic Penn State University (PSU) broth, L. monocytogenes was detected within 24 hours both by plating to MOX agar incubated in air and by a fluorogenic 5' nuclease real-time RT-PCR assay. The RT-5' nuclease polymerase chain reaction (PCR) assay targeting the hemolysin gene (hlyA) detected viable L. monocytogenes directly from the PSU within 24 hours, although a stronger signal was detected after 48 hours of resucitation. The RT-5' nuclease PCR assay bypassed the need for subsequent plating of ground pork to selective agar and thus may shorten the interval to detect low numbers of viable L. monocytogenes following heating of naturally contaminated meat.