Research On The Rapid Detection Method Of Staphylococcus Aureus Based On The New Method Of Chain Exchange Amplification SEA

Staphylococcus aureus(S.aureus)is an important food-borne pathogen.Food poisoning caused by S.aureus enterotoxin occurs frequently.Therefore,it was of great significance to develop a rapid detection method for S.aureus.At present,the detection methods for S.aureus mainly include traditional culture method,immunological detection method and molecular biology detection method.These methods have their own unavoidable shortcomings.For example,the traditional culture method has a long detection cycle and complicated operation;The immunological detection method was low sensitivity;The operation of molecular biology detection method is complex,which is not conducive to rapid detection on the spot.Therefore,it was essential to establish a simple,rapid and sensitive detection method for food-borne pathogenic S.aureus.In this paper,a new detection method of Strand Exchange Amplification(SEA)is constructed.The method adopts the principle of denatured bubble-mediated chain exchange,and can complete the amplification reaction in 1 hour under isothermal conditions.Combined with colorimetric method,only one metal bath was needed to complete the detection of S.aureus.The results were visualized by naked eyes and the detection process was simple.The feasibility,sensitivity,specificity and anti-interference of S.aureus were studied by SEA.The detection limit of S.aureus was as low as 1.0 x 10~-1414 M.In order to further realize the detection ability of SEA detection method for real samples,total nucleic acid(DNA and RNA),DNA and RNA were detected by SEA detection method.The experimental results show that SEA detection method realizes one-step detection of live S.aureus RNA without additional reverse transcription,and the detection efficiency of RNA is higher than that of DNA detection.The detection results can be distinguished by fluorescence interpretation or by the change of system color.In addition,the naked eye colorimetric interpretation results were simple and convenient to operate,independent of professionals and expensive instruments,and the detection results were accurate.The establishment of SEA method is mainly for the detection of actual samples.Therefore,this paper also detects the artificially added pork samples.The results showed that the lowest concentration of SEA was 2 cfu/g contaminated artificially labeled pork products.We further tested 112 meat samples of beef,pork,chicken,dried fish and ham sausage in the market.The results showed that SEA method was compared with traditional culture method.Positive rate was consistent with the traditional culture method.It shows that SEA was a reliable and practical method for the detection of S.aureus in real samples.In this paper,a new one-step RNA isothermal amplification technology platform was constructed,which makes the whole detection process simple,fast,equipment-free and visualized.SEA detection method can also be integrated into microfluidic chip to realize sample diagnosis in a single device,and provide a real-time detection platform for other foodborne pathogenic bacteria in food.