Establishment Of The Detection And Typing Method For Signature Virulence Gene Stx From Shiga Toxin-Producing Escherichia Coli

Shiga toxin-producing Escherichia coli（STEC）is one of food borne pathogens,it has highly pathogenic and the clinical diseases includes diarrhea,hemorrhagic colitis,and the hemolytic-uremic syndrome which could be threat to life.STEC is widely found in the environment,and food is easily infected STEC during preparation,packaging and transportation.The establishment of a fast and simple method for the detection and typing of STEC will be helpful to conduct food safety detecton,and further reduce the risk of STEC infection.In this study,we used Asymmetric Polymerase Chain Reaction（aPCR）combined with Immunochromatography Assay（ICA）to rapidly detect and type the maker toxin gene stx of STEC.The main content and results are as follow:Firstly,a method for rapid detection of the maker toxin gene stx of STEC was established.Biotin marked single stranded DNA of stx1 and stx2 were prepared by aPCR,the amplification products would bound with the upstream primers of the stx1and stx2 gene which were labeled with red polystyrene microspheres,and the conjugates with biotin were specifically bound with streptavidin on the ICA test strip with a red test line.The detection conditions were optimized and the results were shown:The best concentration addition ratio of stx1F to Biotin-stx1R was 1:7,and the ratio of stx2F to Biotin-stx2R was 1:4.The best cycle times of stx1 and stx2 aPCR was55.The best annealing temperature of aPCR was 55℃.The primers addition was 3.0μL(n_Biotin-stx1R=n_Biotin-stx2R=2μmM).The blocking buffer was BSA,and the addtion of the BSA（1%）was 20μL.The STEC containing stx gene could be accurately detected by this method.Then,a method for typing of stx1 and stx2 was established.The digoxin marked single stranded DNA of stx1 and biotin marked single stranded DNA of stx2 were prepared by duplex aPCR.The amplification products could combine with the upstream primers of the stx1 and stx2 gene which were labeled with red polystyrene microspheres,anti-digoxin antibodies and streptavidin were sprayed on the ICA test strip as two different test lines,the conjugates with digoxin or biotin could be captured by the strips.According to color change between the two test lines,stx1 and stx2 could be typed.The detection conditions were optimized and the results were shown:The best concentration addition ratio of stx1 and stx2 duplex aPCR primers stx1F:Digoxin-stx1R and stx2F:Biotin-stx2R were 1:6,the cycle times was 45,the annealing temperature was 59℃,the primers addition was 3.0μL(n_{Digoxin-stx1R}=n _Biotin-stx2R=2μmM).The optimized blocking buffer was 50μL of Seablock.We used this method to detect STEC with different stx genotypes,and the results showed that stx1 and stx2 genes could be accurately classified by this method.Finally,twenty strains were detected and typed by this method.The results showed that the method could accurately detect and type the STEC with different maker toxin gene stx,and showed a negative detecton for non-shiga toxin-producing strains.The method established in this study had the advantages of high selectivity,short time,accurate and safe.The results analysis was convenient and intuition.This method could realized the purpose of rapid on-site detection,it could be applied for the basic laboratory.