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

• DOI: 10.19026/ajfst.5.3200
• Publication Date: 2017-09-15T10:37:53Z
• Authors: Dayong Wang; Zhendong Fang; Chaoxin Xie; Yutong Liu
• Keywords: 0301 basic medicine; 03 medical and health sciences; 3. Good health

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 4×10 9 to 4×10 1 and PCR threshold cycle (Ct) with a correlation coefficient of 0.992 (R 2 = 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.