Development Of Colloidal Gold Strip For Detection Of Listeria Monocytogenes

Listeria monocytogenes distributes widely in the natural environment and can cause the contamination of such food as milk, meat, sausages, vegetables, seafood and fish products. The contaminated food eaten by people can cause gastroenteritis, meningitis, pneumonia, septicemia and abortion in pregnant women, which consequently lead to serious harm and high mortality. Therefore, LM is considered to be one of important foodborne pathogens with high public health significance. It is essential to strengthen the surveillance of LM in food. In this study recombinant LM InlA protein was used as an immunogen to immunize BALB/c mices to prepare the LM monoclonal antibodies. And inactivated LM was used as an immunogen to immunize New Zealand white rabbits to prepare the polyclonal antibody. The monoclonal antibodies were labeled by gold as capturing antibodies and the polyclonal antibody was used as detection antibody to make colloidal gold strip for rapidly detecting LM. The immunomagnetic beads were coated with monoclonal antibody to enrich LM of samples and were evaluated by PCR method.1. Preparation of recombinant InlA proteinThe genomic DNA of LM model strain was used as a template and the full-length inlA gene was amplificated by PCR; After analyzing the antigen epitope, down-stream1200bp fragments of good immunogenicity were amplificated by secondary PCR and the target gene was cloned. Prokaryotic expression plasmid pET-32a-InlA was constructed and induced to express the65kDa target protein in E.coli BL21. The inclusion bodies were purified by nickel column affinity chromatography method to obtain a relatively pure recombinant InlA protein.2. Preparation of InlA protein monoclonal antibodiesRecombinant InlA protein was used as an immunogen to immunize BALB/c mices, Two specific hybridoma cell lines4B4and4E1were screened, which could secret monoclonal antibody against InlA protein. The ascites were purified and the monoclonal antibodies were obtained. The two mAbs had no cross-reactivity with E.coli O157:H7, Salmonella typhimurium, Staphylococcus aureus and non-LM Listeria spp.. The antibody subclasses were IgG1subtype; The antibody titers were1:6.4×104.3. Preparation of polyclonal antibodies of LMHeat-inactivation LM was used as an immunogen to immunize New Zealand white rabbits for preparing the polyclonal antibodies. And the antibody titer was1:32000. The polyclonal antibody was cruded with saturated ammonium sulfate and was further purified with Sephadex G-200.4. Development of colloidal gold strip for detection of LMThe colloidal gold strip for detecting LM was developed by using the gold-labeled monoclonal antibody4B4as capturing antibody and using the polyclonal antibody as detection antibody. The sensitivity of the test strip was2.4×105CFU/mL. The specificity of colloidal gold strip was good, which did not react with L.innocua, L.grayi, Streptococcus, Enterococci feces, Salmonella typhimurium, EHEC O157:H7, only had weak cross-reactivity with Staphylococcus aureus. The results could be observed only in5-10min. The detection limit of analog samples was4.0×106CFU/mL, and the storage period could reach more than120d at4℃.5. Preparation of immunomagnetic beadsThe Fe3O4magnetic particles were synthesized by chemical co-precipitation method and were coated with1%of the acetic acid dissolved chitosan. Immunomagnetic bead was prepared by using250μg/mL monoclonal antibody and1%of glutaraldehyde cross-linking, and was blocked with10%skim milk powder. The efficiency of immune bead was evaluated by PCR method and the results showed that immunomagnetic beads was suitable for enrichment of LM and did not affect the PCR reaction.