Rapid Detection Of Vibrio Parahaemolyticus In Seafood By Loop-mediated Isothermal Amplification

Along with the rapid development of economy, there is more and more public attentionon the food safety. Vibrio parahaemolyticus, as the most common food-borne pathogens ofmicroorganisms, has been concerned by public. However, the current traditional detection aretime-consuming and inefficient, which have not already satisfying people’s high requirementsfor food safety. So, we need develop more efficient and faster detection to monitor V.parahaemolyticus from food, and guarantee the health of human beings.In2000, Notomi T. and his colleagues developed a novel nucleic acid amplificationtechnology which was noted as Loop-mediated Isothermal Amplification (LAMP). In thisstudy, we developed a rapid and sensitive tlh-based LAMP assay on a real-time turbidimeterplatform and fluorescent dye for the detection of V. parahaemolyticus in sea food. The mainresearch contents and the results are as follows:(1) According to conservative gene tlh, which is poisonous force gene ofV.parahaemolyticus based on references. We designed specificity primers for detection ofV.parahaemolyticus, established rapid LAMP method. The results showed that the methodestablished could finish detection for V.parahaemolyticus in just1h with constant65degreesCelsius temperature conditions. The using of turbidimeter could help to observe the wholeprocess of amplification real-timely, and adding SYBR Green I fluorescent dyes could makethe detection results observation fast and convenient; then we experimented with a newformula for dyes. Instead of SYBR Green I, Calcein could make the positive result green so asto much distinct from the orange negative result, Using Calcein, the entire genetic testingprocess could proceed in airtight environments.(2)In this study, we developed a rapid and sensitive tlh-based LAMP assay and apply it inactual operation. The sensitivity of LAMP assay was8CFU per reaction tube in pure culture,80CFU per reaction tube in artificial contaminated oyster. Furthermore, compare to theChinese National standard GB/T4789.7-2008, the LAMP assay combined with3hourenrichment was100%match ratio when applied in70sea food samples.This rapid, accurate, and simple LAMP method may present a useful tool for detectingand quantifying V.parahaemolyticus in food.