The development of rapid real-time PCR detection system for Vibrio parahaemolyticus in raw oyster

Abstract

AIMS: To develop a new rapid real-time polymerase chain reaction (PCR) based detection system for Vibrio parahaemolyticus (V. parahaemolyticus) applicable to raw oyster samples. METHODS AND RESULTS: V. parahaemolyticus cells were artificially inoculated to oysters. Samples were homogenized in 100 ml of sterile saline water and serially diluted to 1.5 CFU ml(-1) level. One millilitre of diluents was centrifuged and the pellet was resuspended with 100 microl of de-ionized water. DNA was extracted by boiling for 20 min, and 0.5 microl was used as a template for PCR reaction. Real-time PCR was performed with TMC-1000 system (1 microl PCR system). The detection system was found to achieve detection limit of 1.5 CFU g(-1) for V. parahaemolyticus. Furthermore, the specificities of these assay systems were confirmed with more than 20 bacterial strains, including various Vibrio species. CONCLUSIONS: Rapid and sensitive food-borne pathogen detection techniques for V. parahaemolyticus is important to the food industry and consumers. The direct detection of V. parahaemolyticus from food is possible with micro real-time PCR system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that oyster samples can be tested for V. parahaemolyticus with a rapid, specific and simple procedure.