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


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Abstract

AimsTo 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 ResultsV. 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 μl of de-ionized water. DNA was extracted by boiling for 20 min, and 0·5 μl was used as a template for PCR reaction. Real-time PCR was performed with TMC-1000 system (1 μl 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.ConclusionsRapid 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 StudyThis study shows that oyster samples can be tested for V. parahaemolyticus with a rapid, specific and simple procedure.

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