Study On Simultaneous And Rapid Detection Of E.coli O157:H7 And Salmonella Typhimurium In Food

For simultaneous detection of Escherichia coli O157:H7 and Salmonella typhimurium in food samples, specific and sensitive monoclonal antibodies and IgY polyclonal antibodies were developed and characterized. Using obtained antibodies, immunoassays were developed for the simultaneous detection of Escherichia coli O157:H7 and Salmonella typhimurium. The results were as follows:1. For the production of antibodies specific to Escherichia coli O157:H7 or Salmonella typhimurium, two kinds of antigens, including heat-killed (HKE, HKS) and formalin-killed (FKE, FKS) whole cell antigens, and gunidine-HCl extracted crude cell surface proteins (GCSPE, GCSPS) antigens were prepared.2. With the guanidine-HCl extracted crude cell surface proteins of Escherichia coli O157:H7 imunuogen, 19 hybridomas that showed positive ELISA reaction with Escherichia coli O157:H7 were obtained after two times cell fusion. After specificity and titer test, 1D11 MAb that produced by 1D11 hybridomas showed the highest titer and specificity to Escherichia coli O157:H7 and the titer of purified 1D11 MAb was 1:25600.3. With the guanidine-HCl extracted crude cell surface proteins of Salmonella typhimurium imunuogen, 12 hybridomas that showed positive ELISA reaction with Salmonella typhimurium were obtained after two times cell fusion. After specificity and titer test, 2G6 MAb that produced by 2G6 hybridomas showed the highest titer and specificity to Salmonella typhimurium and the titer of purified 2G6 MAb was 1:12800.4. After immunized hens with whole cell immunogns,IgY polyclonal antibodyes reacted to Escherichia coli O157:H7 and Salmonella typhimurium were obtained, respectively. The specificity and titer of IgY polyclonal antibodies that isolated and purified by water dilution-ammonium sulfate precipitation method, were tested. The results showed that IgY polyclonal antibodies were specific and the titers were 1:3200, respectively.5. Using monoclonal and IgY polyclonal antibodies, immunochromatography assays for detection of Escherichia coli O157:H7 and Salmonella typhimurium were developed, respectively. The immunochromatography test strips has a detection limit of 105 CFU/ml for Escherichia coli O157:H7 and Salmonella typhimurium, respectively.6. Using colloidal gold labeled monoclonal antibody 1D11 and 2G6 as capture antibodies and IgY polyclonal antibodies as test antibodies, immunochromatography assay for simultaneous detection of Escherichia coli O157:H7 and Salmonella typhimurium in food samples was developed. The simultaneous detection immunochromatographic assay has a detection limit of 105 CFU/ml and the detection limit can be decreased to 100 CFU/ml with 10 h enrichment. Therefore, the rapid immunoassays developed in this study should be very useful tools for rapid screening of Escherichia coli O157:H7 and Salmonella typhimurium in food samples.