Study On Rapid Isothermal Detection Of Nucleic Acid Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus,is one of the most important pathogen in marine food and pickleded food with high salt.If people eat food polluted by V.parahaemolyticus,they may suffer from abdominal pain,vomiting,and even dehydration and death.Therefore,it is urgent to develop a simple,rapid and accurate method for V.parahaemolyticusdetection.Here,toovercomethedisadvantageofV.parahaemolyticus detection by traditional culture method and PCR technology,two nucleic acid isothermal amplification methods,helicase depended amplification（HDA）and denature bubbles-mediated strand exchange amplification（SEA）were studied.Firstly,the HDA method was explored to detect V.parahaemolyticus.The HDA relies on one expensive helicase to open the DNA helix,which greatly hinders the popularization and application of the method.In this article,we performed the cloning,expression and purification of this thermostable helicase to reduce the cost of HDA reaction.The purified helicase,however,showed no activity.So the IsoAmp II Universal tHDA kits（NEB）was used in this study.This method successfully detected V.parahaemolyticus even with a complicate matrix and displayed good specificity.In addition,comparing to other amplification methods,this method can achieve rapid detection V.parahaemolyticus by fluorescence detector within 20 min and by the common water bath pot within 60 min.The big disadvantage of this method is that detection limit is higher than others,even in the presence of melting agents,such as PEG-200 and ET SSB.In order to overcome these shortcoming of the HDA method,SEA method was used to detect V.parahaemolyticus.SEA relies on the breath of DNA duplex and double strands of DNA could reach a dynamic dissociation state.Then the two primers could invaded to the bubble and realize a three-step cycling with only one DNA polymerase.To reduce the detection limit of the original experimental system,the reaction conditions was optimized.The result showed that the method can detect1.0×10^-13M of genomic DNA of V.Parahaemolyticus.Besides this method has a good specificity and strong capacity of anti-interference.Compared with other methods,the reaction of SEA could be finished within 20-40 min.More importantly,SEA can directly detect bacterial liquid of V.parahaemolyticus,avoid the tedious steps of extracting genomic DNA and the requirements of professional operator,greatly shorten the detection time.By using a colorimetric reagent,visual detection of V.parahaemolyticus could be achieved,which get rid of large equipment and can be realized by simple thermos cup or metal bath.Eventually,actual sample was successfully detected by SEA method.In this article,the optimized SEA method could meet the requirement of rapid and on-site detection and have a significant role in the area of instant detection,animal disease monitoring and food safety.