Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39–100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 10¹, 10², and 10³ CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.

Seafood mislabeling threatens customer rights and causes economic loss worldwide. The information on seafood misrepresentation in Thailand is still lacking, and the investigation and monitoring program must be well established. This study investigated the mislabeling status of imported seafood in Thailand using the DNA barcoding technique. A total of 45 imported seafood products from five distributors were included. Scientific, common, local, and market names of seafood samples were obtained from FAO and Fishbase databases. DNA was extracted, and PCR was performed using a universal primer targeting the COI gene. Species of each sample were identified with over 98% similarity based on COI sequence analysis. DNA sequence revealed 11 mislabeled samples. Among substituted species, Pangasianodon hypophthalmus and Thunnus maccoyii were found to be endangered species according to IUCN status. Products obtained from Brand-C showed the highest mislabeling rate (42.85%). The phylogenetic analysis adopted with the TIM2+F+I+G4 model showed the sequenced DNA similar to the NCBI database reference sequence. Overall, mislabeled products of imported seafood were found at the rate of 24.44%, suggesting that strict surveillance for seafood substitution should be implemented in Thailand.