Construction of method for rapid detection of Vibrio Parahaemolyticus using the quantitative real-time PCR based on the ToxR gene

Abstract

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 primer-dimers 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 4×109 to 4×101 and PCR threshold cycle (Ct) with a correlation coefficient of 0.992 (R2 = 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. © Maxwell Scientific Organization, 2013.