parahaemolyticus from other Vibrio

parahaemolyticus from other Vibrio species and non-Vibrio bacterial strains. This PCR assay produced a 369-bp fragment, and the sensitivity was 0.17 pg purified genomic DNA from V. parahaemolyticus. Kang et al.[13]established a two-step ultrarapid real-time PCR assay with a microchip to detect V. parahaemolyticus carrying the tdh gene. This method used 6-μL reaction volumes and total run times of about 6 min. To the best of our knowledge, this is the most rapid detection approach thus far reported. Copin et al.[14] evaluated a PCR assay using most-probable number enrichment cultures for the detection of total and pathogenic V. parahaemolyticus in frozen shrimp. The detection limit was one cell per gram and can be expected to be completed within two days. 3.2 In the present study, a highly specific nested PCR assay was established to immediately detect V. parahaemolyticus in food samples, targeting the VP1331 gene, which was aligned as the specific gene of V. parahaemolyticus. The results show that this assay could detect V. parahaemolyticus in both pure culture broth and artificially infected seafood samples, and the detection limit was 10 fg with purified DNA and 6.6 CFU with pure culture. As shown in this study and a previous report[11], this nested PCR detection limit is lower than that of conventional PCR. This nested PCR protocol can be completed in about five hours, including half an hour for sample processing, two hours for bacterial enrichment, two hours for PCR, and half an hour for electrophoretic analysis. Therefore, the nested PCR assay is relatively time efficient. The number of nested PCR cycles from 30 to 27 and 25 to save time, but no bands were detected; thus, 30 cycles were determined to be optimal to avoid non-specifically amplified bands. As the results show in the detection of one hundred specimens purchased from a local supermarket, the isolation rate between nested PCR and conventional PCR was different and the nested PCR showed higher sensitivity and accuracy. In conclusion, this nested PCR can be invoked as a robust tool for the detection of V. parahaemolyticus in seafood.