Duplex electrochemical biosensor for the detection of the tdh and trh virulence genes of Vibrio parahaemolyticus in oysters

Vibrio parahaemolyticus is a bacterium present in estuarine environments. Since the first outbreak in Japan in 1950, it has been a dominant cause of foodborne infections throughout the world. Vibrio parahaemolyticus strains can accumulate in shellfish and cause gastroenteritis. The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. Conventional methods used for V. parahaemolyticus monitoring in seafood, based on microbiological counts, are time consuming and laborious, and they cannot identify virulence genes. Therefore, a duplex electrochemical biosensor for the detection of the tdh and trh genes of V. parahaemolyticus in oysters was developed. Oyster homogenates were prepared, enriched for bacterial content, and DNA was extracted. Then, a duplex PCR with tailed primers was performed to amplify both genes. Afterwards, sandwich hybridisation assays were carried out on magnetised working electrodes of an array. The electrochemical biosensor was able to detect the tdh and trh genes from V. parahaemolyticus strains, and no cross-contaminations were observed between electrodes. The limit of detection for both genes was 10 pg/μL and no cross-reactivity was observed when using other Vibrio species and non-Vibrio pathogens. The biosensor was able to detect as low as 1 CFU of V. parahaemolyticus in oyster homogenate. Screening of oysters from an infectivity experiment and comparison with other techniques proved the proper performance of the biosensor and its applicability to the analysis of natural samples, with added advantages of specificity, duplexing capability, portability and provided virulence information.