Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed forVibrio vulnificus hemolysin A gene (vvhA ) for TaqMan-based real-time PCR method enabling specific detection ofVibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for allV. vulnificus isolates, also exhibited positive cycle threshold (CT ) values for otherVibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 103 V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification ofvvhA . Detection of 3 × 103 CFUV. vulnificus , resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection ofV. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe onvvhA for TaqMan-PCR-based detection ofV. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducingV. vulnificus -related illnesses and deaths.