Construction of Method for Rapid Detection of Vibrio Parahaemolyticus Using the Quantitative Real-Time PCR Based on the ToxR Gene

In this research, a sensitive, rapid and highly reproducible SYBR green based real-time PCR assay was developed for detection of toxR positive pathogenic Vibrio parahaemolyticus. To establish a real-time PCR assay for accurate and rapid detection of Vibrio parahaemolyticus. The special target sequence of toxR gene of Vibrio parahaemolyticus was amplified and characterized with a pair of primes. Reaction system and determination approach of real-time PCR were established for detection of Vibrio parahaemolyticus. The foodborne pathogen of Staphylococcus aureus, Salmonella were used as specificity reference for the method, the amplification curves were observed as a typical “S” curve, other pathogens were also tested and no amplification was observed. In addition, the results of melting curve analysis showed only a specific peak with a melting temperature of 86.33°C and no primerdimers peak was observed. These findings indicated that the PCR primers had high specificity. Analysis of standard curves revealed excellent correlation between the number of copies (in the range of 4×10 to 4×10 and PCR threshold cycle (Ct) with a correlation coefficient of 0.992 (R = 0.992). It was found that the limit of this assay was 15 CFU/mL for pure culture and 1.535pg for genomic DNA. The total detection assay could be completed in 2 h. Results indicated that real-time PCR detection methods established in this research was accurate, sensitive, rapid, reproducible for the quantitative detection of environmental Vibrio parahaemolyticus.