| Enterohemorrhagic Escherichia coli (EHEC) is an important intestinal pathogenic E. coli and zoonotic pathogen, which mainly causes hemorrhagic colitis in humans. O157:H7as a crucial serotype of EHEC is a pathogen responsible for many scattered or epidemic diarrhea cases in the world. EHEC O157:H7is foodborne pathogen for it infected people primarily through consumption of contaminated foods. The risk of O157:H7infection is higher than those of other pathogens because its infection dose is much lower. Therefore, the detection of EHEC O157:H7is of great significance. However, the bacterial isolation and identification is time-consuming and complicated in operation, and has certain requirements for both apparatus and operator similar to other laboratory detection methods. So the method is not suitable for the detection at point-of-care in low resource settings and massive samples. This study combined with Colloid Gold Immunochromatographic Technology using two specific monoclonal antibodies to establish a sensitive, specific, fast and simple method for detection of EHEC O157:H7.1. Screening of EHEC O157:H7monoclonal antibodyScreening from eight specific hybridoma cell lines secreted specific monoclonal antibodies against EHEC O157:H7O-antigen prepared in the lab by using ELISA test, two cell lines1H3and6F4were selected to have minimum influence on antigen recognition epitopes.1H3and6F4were identified to belong to IgG and IgM subtype, respectively. Antigen recognition epitopes of two McAb were proved to have little interaction effect by using competitive ELISA composed of6F4McAb marked on horseradish peroxidase(HRP) and1H3. Titers of two McAb were both above1×105, which could be used for the preparation of lateral flow test strip of sandwich method.2. Resuscitation of hybridoma cell strains and preparation of monoclonal antibodiesTwo hybridoma cell strains were resuscitated and injected in the abdomen of BALB/c mice for producing McAb.1H3McAb was purified by octanoic acid and ammonium sulfate precipitation method, and6F4McAb was extracted by ammonium sulfate and was purified by the Sephadex G-200column. The concentration of1H3and6F4were2.58mg/mL and1.00mg/mL, respectively.3. Development and Application of Colloidal Gold Test Strip1H3conjugated with gold nanoparticle was dispensed onto the glass fiber membrane;6F4was coated on the detection line of nitrocellulose membrane; rabbit anti-mouse IgG was coated on quality control line of the rear end of nitrocellulose membrane. Then the lateral flow test strip was assembled to detect EHEC0157:H7, eleven kinds of E. coli non-0157:H7, Staphylococcus aureus, Salmonella typhimurium, and Listeria monocytogenes. EHEC O157:H7showed positive result and other strains showed negative results, indicating that the specificity of this test strip was good. The detection limit was1×105CFU/mL of EHEC O157:H7pure culture. After enriching for6h, the sensitivities were lOCFU/mL-100CFU/mL of artificially milk or pork samples contaminated by EHEC O157:H7.4. Preliminary application of lateral flow test strip200pork samples collected from the supermarket were detected after enriched in modified EC broth for12h. The enrichment broth was heated for30min at100℃of water bath and was detected by EHEC O157:H7lateral flow test strip. The results showed that only one sample was positive. Meanwhile, the conventional culture method prescribed in national standards and duplex PCR method developed by our lab were used to detect the EHEC O157:H7in the samples. The coincidence rates of three methods were100%. |
PDF Full Text Request