Development of Duplex PCR for Vibrio parahaemolyticus Based on bla_CARB-17 and toxR Genes

A duplex polymerase chain reaction(PCR)method was established to simultaneously detect the bla_CARB-17 and toxR genes of Vibrio parahaemolyticus. Two sets of primers were designed and synthesized according to the bla_CARB-17 and toxR gene sequences of Vibrio parahaemolyticus. PCR amplification was performed in the same reaction system. The reaction parameters(annealing temperature,annealing time and primer ratio)were optimized to determine specificity and sensitivity. The isolates of Vibrio parahaemolyticus in our laboratory were detected by this duplex PCR. The results showed that the best annealing condition of the duplex PCR was 52℃ 30 s,and the ratio of primers was 1:1.Using two pairs of primers,303 and 350 bp fragments of bla_CARB-17 and toxR genes were amplified,respectively. No amplification was observed in other control bacteria,indicating that this method had good specificity. The minimum detection limit of duplex PCR was 1×10² CFU/mL,and all the 56 isolated of Vibrio parahaemolyticus were positive for detection. The established duplex PCR method is simple,efficient,rapid,and specific. It is suitable for the detection of large-scale samples of Vibrio parahaemolyticus.