Detection of Vibrio parahaemolyticus in seafoods by duplex real-time quantitative PCR targeting genes tlh and tdh.

To develop a duplex real-time quantitative PCR assay for Vibrio parahaemolyticus (V. parahaemolyticus) detection in seafood,primers and TaqMan probes targeting species-specific gene tlh and virulence gene tdh were selected and the specificity and sensitivity were then tested. 50 samples collected from food markets in Zhoushan were screened for V. parahaemolyticus by duplex real-time quantitative PCR as well as the regular PCR. Only V. parahaemolyticus strains exhibited typical curves,and when other bacteria concurred with V. parahaemolyticus in seafoods,they were not amplified and would not interfere the results of the detection. The detection limitation of this method was from 49.8pg (FJ14) to 77.8pg (BJ97) for purified genomic DNA,and from 56 (FJ14) CFU to 371(BJ97) CFU per ml for pure cultures. 32 out of the 50 samples were positive for tlh and 3 were positive for tdh. Although the results were consistent with the conventional method,this assay was more rapid and reliable for detection of V. parahaemolyticus.