Development Of An Immunoassy For Cronobacter Sakazakii

Cronobacter sakazakii is a kind of opportunistic pathogen. C. sakazakii infections can lead to severe disease manifestations such as necrotizing enterocolitis, meningitis, brain abscesses and bacteremia in Neonates, infants and the elderly with high fatality rate.Therefore, it is significant to develop rapid detection method for Cronobacter sakazakii.In this study, based on 59 immune proteins identified already in 8 strains of C.sakazakii (CICC22922, CICC21560, 90905, ATCC29004, ENS6607, ATCC12868,ENS70307-2, ENS07208), two outer membrane proteins with high specificity were selected.Specific parts of these two proteins were determined based on protein sequences comparison and the three-dimensional structures of protein A and potein B. Due to the seleted gene parts were too short, two repeated fragments of the two gene were ligated by two flexible peptides, respectively. The sequences were synthesized and ligated into vector pUC57. The recombinant pET-26b(+)expression plasmid was constructed by double digestion and ligation. The proteins were recombinantly expressed in E. coli BL21 and expression conditions were optimized. Fusion protein A has a molecular weight of about 22 kDa, while fusion protein B has a molecular weight of about 20 kDa.The expressed proteins were purified with immunoaffinity chromatograpHy and used to immunize New Zealand white rabbits and SPF hens to prepare polyclonal antibodies.The ELISA method was established and optimized. The chicken polyclonal antibody against protein A (A-IgY) was used as the capture antibody with coating amount of 0.5 ?g,while the rabbit IgG against protein A (A-IgG) was used as the detection antibody with a 200 folds dilution. Blocking solution is 1% skim milk powder in PBS, while enzyme-labeled secondary antibody was diluted 20,000 folds. Detection capacity analysis showed that the detection limit was 1.5 × 107 CFU/mL and the limit of quantification was 3.9 × 107 CFU/mL. The intraplate variation was 1.69%-4.62% and the interplate variation was 4.12%-7.4%. Three different strains of Cronobacter sakazakii, 5 different species of Cronobacter and 6 strains of non- Cronobacters were detected by this method. The results showed that the detection method had good specificity.