Study On A Novel Immunoassay For V.parahaemolyticus

Food-borne Disease is one of the most common and widespread diseases in the curre ntlyworld. The food-borne disease caused by pathogenic bacteria is an significant public healthsecurity problem at home and abroad. Along with economic development, the food poisoningcases caused by eating aquatic product polluted by Vibrio Parahaemolyticus has topped thelist of microorganism food poisoning in a lot of areas especially coastal regions in China.There are respective advantages and disadvantages in each of the methods has been reportedfor V. parahaemolyticus detection. Therefore, it is necessary to continuously explore a novelmethod which is more convenient, efficient and sensitive. The immunoassay which is mostwidely used was improved in this research. We prepared three kinds of polyclonal antibodyprobes specific to surface proteins of V. parahaemolyticus. Then combined with magneticseparation and upconversion nanoparticles probe technology, developed a novel detectionmethod of V. parahaemolyticus.Firstly, The complete flagellin gene （FlaA）, two kinds of outer membrane proteingene （OmpU and OmpW） from V. parahaemolyticus ATCC1780were amplified. Next,the genes were cloned into pET28a、pET24a plasmid vectors, and then the recombinant plasmid （pET28a-FlaA、pET24a-OmpW、pET24a-OmpU） were transferred into E. coli BL21（DE3）. After that, the genes coded proteins were expressed by IPTG and purified using Ni-NTA resin affinity chromatography. A clear single target band of the antigen protein was proved by SDS-PAGE.Secondly,2rabbits a team were immunized by inactived V. parahaemolyticus, purified FlaA, OmpU and OmpW proteins repectively. The obtained four kinds of polyclonal antibodies were purified by saturated ammonium sulfate method. The titers of polyclonal antibodies above were1:160,000,1:16,000,1:16,000,1:8,000approximately aferpurified. Whats more, in order to further quantitative evaluate the specificity of the antibodies, Flow cytometry was employed. Measured binding fraction of V. parahaemolyticus and antibodies are:91.57±5.40%,89.77±6.89%,34.32±2.78%, and63.53±5.40%.Thus, anti-V. parahaemolyticus antibody and anti-FlaA antibody were employed forsubsequent experiments.Thirdly, amine-functionalized Fe3O4magnetic nanoparticles（MNPs） were synthesized by Hydrothermal-Solvothermal method. Assembled with anti-V. parahaemolyticus antibody into immunomagnetic beads when the optimal initial concentration of antibody solution is600μg/mL, incubating time is40min. Meanwhile, amine-functionalized NaYF₄:Yb,Er upconversion nanoparticles（UCNPs） which are stable, sensitive and no autofluorescence were synthesized by Hydrothermal method. Assembled with anti-FlaA antibody into immunoUCNPs when the optimal initial concentration of antibody solution is800μg/mL, incubating time is60min. After incubating with these two conjugation, asandwich-type immunoassay format could be determined by analysis its characteristicflurescence peak intensity. The detection limit for V. parahaemolyticus was10³cfu/mL,and the linear range was from5×10³to5×10⁵cfu/mL（Y=178.25X-506.41, R2=0.991 9）. In simulated samples inspection of aquatic products, the recovery was90.7%-96.7%, consistent with the traditional plate count method.