| With the improvement of people’s living standards and the increasing variety of food,food safety issues have attracted more and more attention.Vibrio parahaemolyticus(V.parahaemolyticus)and Salmonella are two of the most important food borne pathogenic microorganisms that cause seafood poisoning.Due to their high pathogenicity and high mortality,these two pathogens have posed serious threats to people’s life and health.Therefore,the development of a simple,rapid and sensitive on-site detection method for V.parahaemolyticus and Salmonella not only provides a technical platform for point of care testing(POCT)in the field of molecular biology,but also provides a new and effective method in inspection and quarantine,which has significant practical meaning for improving people’s food safety and health.In order to achieve the purpose of rapid and effective detection of V.parahaemolyticus and Salmonella on site,we compared various approaches for V.parahaemolyticus and Salmonella detection based on the development of advanced food borne pathogens detection technologies.By summarizing and analyzing the advantages and the disadvantages of each method,we adopted a shorter time-consuming method for detection of nucleic acid of V.parahaemolyticus and Salmonella.Denatured bubble-mediated Strand Exchange Amplification(SEA)is based on the spontaneous respiration of double-stranded DNA template to generate denatured bubbles.The primers are embedded into the denatured bubble and extended under the action of Bst DNA polymerase to form a large number of exponential amplification products in a short time.In this study,the visual detection of V.parahaemolyticus can be realized within 1 hour by using this technology platform and colorimetric dyes.The whole detection process can be completed by using a small metal heating module instead of sophisticated instruments.After a pure culture of V.parahaemolyticus in 3%Na Cl nutrient broth,the detection limit was 1.0×10~3CFU/m L.This technology show great application potential for sample-to-answer,and can provide strong technical support for environmental monitoring,food hygiene,and disease diagnosis.However,the sensitivity of SEA method was relatively low,and the whole reaction took long time,indicated some limitations in the POCT of food-borne pathogens.In order to meet the requirements of POCT detection of V.parahaemolyticus and Salmonella,our group improved SEA technology and developed an Accelerated Strand Exchange Amplification(ASEA)based on narrow variable temperature.ASEA technology has the advantages of simple system,fast response,and higher sensitivity.This technology can obtain more denatured bubbles and single strands by introducing repeated narrow thermal cycles,and shorten the time of each cycle due to smaller temperature changes,thereby greatly improve the sensitivity and speed of the reaction.At the same time,it is equipped with a new nucleic acid release agent,and the entire detection process can be completed within30 minutes.The detection limits for V.parahaemolyticus and Salmonella were 25CFU/m L and 100 CFU/m L in pure cultures,respectively.The V.parahaemolyticus was mixed with scallop samples and cultured 12 hours,the detection limit of this technique was 400 CFU/m L.Compared with SEA,the ASEA technology greatly shortened the reaction time,improved the detection sensitivity,and realized the purpose of rapid detection of V.parahaemolyticus and Salmonella on site.In short,this paper optimized the SEA technology,and finally realized the rapid detection of V.parahaemolyticus and Salmonella through the ASEA technology.It has the advantages of simple operation,strong specificity and high sensitivity,and realized the POCT nucleic acid detection of foodborne pathogens in seafood.It can solve the problem of real-time nucleic acid detection of food-borne pathogens in seafood.This study suggested broad application value and will provide technical support not only for food safety control,environmental health monitoring,but also for the early diagnosis of diseases. |
