Application of a real-time PCR method for rapid detection of Vibrio vulnificus in oyster

Objective:The aim of this study was to develop a real-time PCR assay for detecting Vibrio vulnificus in raw oysters and evaluate the specificity,sensitivity and accuracy of the assay.Methods:The genomic DNA of sample after 5 h pre-enrichment or without pre-enrichment in 3% NaCl alkaline peptone water was extracted by using two methods(QIAamp DNA mini Kit and boiling lysis assay).Then the primers and probe originated from vvhA gene were designed in order to establish a real time PCR assay.Results:It was a specific assay for Vibrio vulnificus sample,and other bacteria were negative for this reported here.The real-time PCR assay combined with DNA extraction by QIAamp DNA mini Kit is suitable for the detection of Vibrio vulnificus in oyster samples.The detection limit of the method was 1.4 cfu/ml.Conclusion:This study shows that the real-time PCR assay was specific,sensitive and accurate,and it is applicable to rapid detection of Vibrio vulnificus in oyster samples in eight hours.