| Vibrio parahemolyticus(V.parahemolyticus)is one of the foodborne pathogens which widely exists in seafood.With the development of economy and the increasing demand of fresh sea food,and the proportion of food poisoning caused by V.parahaemolyticus was increasing year by year.Therefore,developing a rapid and efficient detection method for V.parahaemolyticus identification is crucial for food safety and disease diagnosis.To realize simple and rapid detection of V.parahemolyticus,loop mediated isothermal amplification(LAMP)was used to detect V.parahemolyticus.The result indicated that the method had a good specificity with the existence disturbance and the detection limit could reached 1.0×10~-1717 M.Besides,the method could directly detect bacteria samples without extra nucleic acid extraction,which greatly reduced the reaction time,compared to the traditional method.Using a colorimetric reagent,the visual detection LAMP.This can greatly simplify the detection process and shorten the time,providing an effective method for on-site rapid detection with low cost and no need of large equipment.In spite of the high sensitivity of LAMP method,it is highly susceptible to pollution due to high sensitivity,which is the biggest obstacle to LAMP application.To solve this problem,we used restriction endonuclease and created a new technology called primer enzyme method to eliminate carryover contamination.The result indicated primer enzyme method can efficiently elimanate the contamination from last reaction product,and the elimanate effect could reach to 3 orders of magnitude,which effectively reduced the false positive of LAMP method and improved the detection accuracy.Based on the above results in eliminating the carry-over contamination in LAMP reaction using primer enzyme method,the present study applied this method to PCR reaction and SEA reaction.The primer enzyme method was modified and optimized for the PCR and SEA method.The results showed that the primer enzymatic method could eliminate the PCR product pollution of 560 ag,and the peak time could be delayed for higher concentration PCR product pollution.As for the SEA reaction,primer enzyme method could also delay the reaction time of contaminated reaction.Although the effect of primer enzyme reaction in eliminating contamination of PCR and SEA reaction was poor,it could provide some new clues for the research of contamination control.In this paper,the detection of V.parahemolyticus by LAMP technology has achieved the purpose of rapid and sensitive detection,and has excellent specificity,sensitivity and anti-jamming capacity.Besides,a novel kind primer enzyme method to control carry-over contamination was proposed and effectively solved the pollution problem in the process of LAMP detection.At the same time,this method was applied to other nucleic acid amplification technology.It is therefore conducive to promote the application of nucleic acid amplification methods in the field of point-of-care testing(POCT)and in vitro diagnostic(IVD). |
