Study Of Rapid Immunological Detection Methods Of EHEC O157:H7

Enterohemorrhagic Escherichia coli O157:H7(EHEC O157:H7)is one of the important foodborne pathogens.The bacteria can contaminate meat,milk and other foods,therefore it has brought serious harm and loss to breeding industry and food industry.The infective dose of the bacterium is extremely low,it can cause hemorrhagic colitis,hemolytic anemia,thrombotic thrombocytopenic purpura in people after infection.What is more,the harm of infected EHEC O157:H7 is more serious to the elderly,children and people with immune deficiency.Therefore,timely detection is an important means to prevent and control O157:H7 infection.Meanwhile,due to the short shelf life of meat,milk and other fresh food,the detection method is required to be as simple and fast as possible while taking into account the sensitivity and specificity.However,the classical selective medium separation identification takes a long time and is easily interfered by false positive.Molecular biological methods such as PCR have high requirements on instruments and equipment,which have certain limitations when used to detect large quantities of food samples.Therefore,high potency and highly specific monoclonal antibodies were used to construct chemiluminescent enzyme immunoassay(CLEIA),gold nanopariticle immunochromatographic strips and lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips(EuNP-ICT)for EHEC O157:H7 detection,which can realize rapid qualitative and quantitative detection of EHEC O157:H7.Test Ⅰ Preparation of McAb anti-EHEC O157:H7 O-SPThe EHEC O157:H7 LPS were prepared by hot phenol-water method.And the complete antigen(LPS-BSA)were prepared by coupling LPS with bovine serum albumin(BSA)by means of carbodiimide method.After immunizing 8 weeks old female Balb/c mice,monoclonal antibody was prepared by hybrid tumor method.Then O-SP(prepared by acid hydrolysis method)and E.coli O157:H7 were used as the coating antigen to screen positive putative hybrids by indirect-ELISA.And tumor induced ascites were used to obtaining McAb.The McAb have no cross-reaction with other nonO0157 foodborne pathogens,and it’s potency is 1:106.The successful preparation of excellent monoclonal antibody has laid a good foundation for the establishment of various immunological rapid detection methods.Test Ⅱ Establishment of a double antibody sandwich chemiluminescent enzyme immunoassay for detecting E.coli O157:H7In order to detect EHEC O157:H7 contamination in milk,rabbit polyclonal antibodies were used as capturing antibodies and a monoclonal antibody against E.coli O157:H7 was used as a detecting antibody.A double antibody sandwich chemiluminescent enzyme immunoassay was constructed for the detection of E.coli O157:H7 by using these two antibodies.The specificity of this method is good,and the detection limit is better than ELISA,up to 2.5 ×104 CFU/mL.In addition,this method has no cross reaction with other E.coli,Salmonella,Listeria,E.sakazakii,etc.In the detection of E.coli O157:H7 in milk samples,the method has a shorter pretreatment time than ELISA.In conclusion,this study provides a highly accurate and specific method for the detection of E.coli O157:H7.Test Ⅲ Establishment of gold nanopariticle immunochromatographic strips for detection of E.coli O157:H7Gold nanoparticles(Au-NPs)were prepared by sodium citrate reduction in this study,and the Au-NPs dispersed evenly with 18nm in diameter under transmission electron microscope(TEM).The McAb anti-EHEC O157:H7 O-SP were marked by Au-NPs,and rabbit polyclonal antibody(PcAb)against EHEC O157:H7 coated on the test line of strip.The specificity of this strip is good,and have no cross-reaction with other non-O157 foodborne pathogens.The the visual limit of detection(LOD)of the strip is 5×105 CFU/mL.Due to the simple operation and low cost,the strip is very suitable for rapid qualitative screening of large quantities of samples.In addition,this study also lays a good foundation for the further development of lanthanide-labeled fluorescent-nanoparticle immunochromatographic stripsbased on the same principle,which can realize quantitative detection.Test Ⅳ Establishment of gold Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips for rapid and quantitative detection detection of E.coli O157:H7In this study,Eu(Ⅲ)chelate nanoparticles(EuNPs)conjugated to the McAb anti-EHEC O157:H7 O-SP were used as fluorescence reporters.The strip was visually observable within 15 min under dark conditions using an ultraviolet light source and a test-strip reader was used for quantitative analysis.Under optimal conditions,the visual limit of detection of the strip was 104 CFU/mL,and the LOD for quantitative detection was as low as 5×102 CFU/mL using the test-strip reader.No cross-reaction of other E.coli strains with different somatic antigens or other bacterial non-E.coli strains was observed,indicating the high specificity of this method.Additionally,the simulated contaminated samples of beef,milk,pork and chicken can be tested postive after preincubating for 8,6,8 and 8 hours,respectively.Futhermore,average recovery of E.coli O157:H7 spiked in pork was 84.5%-101.4%,indicating the accuracy and reliability of the novel test strip.These results indicate that the developed EuNP-ICT strip assay has potential as a powerful tool for monitoring foodborne pathogens in food-safety testing.