Establishment Of An Immunoassy Method For Cronobacter

Cronobacter is a group of foodborne pathogenic bacteria,which can cause neonatal meningitis,bacteremia,osteomyelitis and other diseases.The operation of the traditional detection methods is tedious and complicated,so establishment of a rapid,stable and convenient detection method is of great significance for the prevention and control of Cronobacter.In this study,we screened and prepared monoclonal antibodies based on Cronobacter proteins as immunogens,and established a rapid colloidal gold detection method for Cronobacter.The following two main studies were carried out.（1）The preparation of Cronobacter monoclonal antibodies in mice.Recombinant expression vector was constructed based on two specific genes in Cronobacter.Two recombinant proteins were induced and optimized in prokaryotic expression.Both recombinant proteins were expressed insolubly.Large amount of the target proteins were purified using Ni-NTA Resin to obtain two recombinant proteins.Proteinαand?showed a molecular weight of about 22 k Da and 20 k Da respectively.Two recombinant proteins were used as immunogens for the preparation of Cronobacter monoclonal antibodies in mice,and the antiserum potency of the immunized mice was measured by indirect ELISA.The results showed that highest antiserum titers reached 1:243000.The spleen cells of the mice with high antiserum titers were selected for cell fusion with myeloma cells;After clone and culture,total eight hybridoma cell lines（α1）B-3、（α2）C-9、（α3）E-5、（α3）H-7、（α3）E-8、（β1）B-6、（β2）C-10、（β3）E-8 were obtained.The total proteins of the bacterium were transferred to the membrane for Western Blot analysis,and the results further demonstrated that the prepared monoclonal antibody could specifically identify Cronobacter.（2）A colloid golden test strip for rapid detection of Cronobacter was prepared using the monoclonal antibody.Firstly,20 nm of colloidal gold particles were synthesized,and colloidal gold was coupled with the monoclonal antibody.The optimal conditions for coupling were adding 7μL of K₂CO₃and 10μg of antibody in the reaction mixture.The detection conditions of the colloidal gold test strips were optimized as follows:secondary antibody was diluted by eight folds in C the line;and the detection antibody coating amount was 1.0 mg/m L in T line;Au NPs-m Ab loading volume was 10μL;and loading buffer was Tris-HCl buffer.The sensitivity of the test strip was verified,and the results showed that the detection sensitivity was 10³cfu/m L.Specific verification results showed that the test strip could detect C.sakazakii and most of the species of Cronobacter spp,but could not detect E.coli,Salmonella,L.monocytogenes,S.aureus,P.aeruginosa,showing good genera specificity.Milk powder was selected as the real sample to verify the detection effect of the test strip,and the results showed that when the enrichment time was 6 h,the detection limit of the strip for Cronobacter in the milk powder could reach 1×10⁰cfu/10g.