Study On PCR And Immunology Methods For Detecting Enterohemorrhagic Escherichia Coli O157:H7

Escherichia coli O157:H7 is one species of highly harmful food-borne disease-caused bacteria, which tremendously influenced all countries around the world in the recent twenty years. This study discusses the researches which were involved in microbiologic identification, qualitative and quantitative PCR and immunologic detective technologies.Firstly, microbiologic incubation and detection had been carried out to understand the fundamental biologic characters of E. coli O157:H7 and to elementarily identify it by using SMAC culture medium.Secondly, five pairs of primers had been designed to conduct qualitative PCR detection. The result presented that the primers all could be amplified, furthermore, all had favorable specialty. Optimize examination confirmed the best annealing temperature was 58~60℃, the best concentration of primer was 2μmol/l. The sensibility of regular PCR detection was 10~3cfu/ml. The results of SYBR Green I dye-based fluorescent quantitative Real-time PCR showed a well amplification and its detective limit is 1cfu/ml. The Real-time PCR standard curve equation was built by conducting the optimize examination to Real-time PCR involved in primers and template concentration etc.Thirdly, the polyclonal antibody had been obtained through animal experiment, whose titer was 1:25000, and detective sensibility of ELISA method was 4.17×10~3cfu per hole, without cross-reaction with other E. coli.