Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood

ABSTRACT

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V. parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 10(7) CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.