Detection of total and pathogenic Vibrio parahaemolyticus in shellfish: comparison of PCR protocols using pR72H or toxR targets with a culture method

Abstract

PCR protocols directly applied to enrichment broth cultures were compared with a culture method based on the ISO reference for detection of Vibrio parahaemolyticus in 57 natural bivalve mollusc samples. Comparisons were made on different primer pairs specifically targeting the V. parahaemolyticus-specific toxR gene (Vp-toxR) and pR72H fragment, and also tdh and trh hemolysin genes. The PCR method using these different primer pairs and the culture method were also examined for their limits of detection (LOD). The LODs ranged from 7-24 pg of purified DNA per reaction tube (RT) for primer pair Vp-toxR, but for primer pair pR72H, varied greatly depending on the V. parahaemolyticus strains used (0.7 pg-10.6 ng/RT). The Vp-toxR and pR72H primers allowed the detection of V. parahaemolyticus in 25 and 8 out of the 57 samples, respectively, while only 3 V. parahaemolyticus-positive samples were obtained by the culture method. The effective presence of V. parahaemolyticus in the Vp-toxR-positive samples was confirmed by sequencing the PCR products. The trh and Vp-toxR genes were simultaneously detected in 14% of the samples, which were thus considered as presumptively contaminated with pathogenic V. parahaemolyticus. These results emphasize the need for an efficient survey of both the total and pathogenic V. parahaemolyticus present in seafood in France. The PCR protocol targeting Vp-toxR followed by tdh and trh genes is an efficient and reliable method for the detection of total and presumptively pathogenic V. parahaemolyticus in bivalve molluscs.