Establishment and application of dual real-time PCR for detection of virulence genes of Vibrio parahaemolyticus

ObjectiveA dual real-time polymerase chain reaction (PCR) method for simultaneous detection of two virulence genes tdh and trh of Vibrio parahaemolyticus was established, and the virulence genes carried by 2 771 strains of foodborne Vibrio parahaemolyticus in China were comprehensively detected.MethodsAccording to the tdh and trh virulence genes of Vibrio parahaemolyticus, PCR primers and fluorescent probes were designed respectively, the real-time PCR reaction system and reaction procedure were optimized, and a dual real-time PCR detection method which can detect the two virulence genes at the same time was established. The virulence genes carried by 2 771 strains of foodborne Vibrio parahaemolyticus isolated in 2015 and 2016 were detected by the established method, and compared with the result of PCR method to evaluate the sensitivity, accuracy and specificity of the method.ResultsThe established dual fluorescence PCR method could detect both tdh and trh virulence genes at the same time, and its sensitivity was 1.5×10-4 ng/μL. The accuracy and specificity were 100%. In 2015, the carrying rates of tdh and trh genes in foodborne Vibrio parahaemolyticus in China were 0.26% (3/1 137) and 1.67%(19/1 137) respectively, and were 0.24%(4/1 634) and 0.43%(7/1 634) in 2016 respectively.ConclusionIn this study, a dual real-time PCR method for simultaneous detection of tdh and trh virulence genes of Vibrio parahaemolyticus was established, which could quickly and accurately screen the virulence genes of Vibrio parahaemolyticus. The carrying rate of tdh and trh virulence genes of Vibrio parahaemolyticus isolated from food in China was low. The dual real-time PCR method can be applied to the study of the pathogenicity of Vibrio parahaemolyticus in food, and provide scientific data for the risk assessment of dietary exposure to Vibrio parahaemolyticus in China.