运用抗原芯片原理与竞争免疫层析技术制备鱼类病原性溶藻弧菌快速检测试纸  

Development of a Competitive Gold Immunochromatography Test Strip for Rapid Detection of Vibrio alginolyticus in Fish

作　　者：李嘉文 绳秀珍[1] 唐小千[1] 邢婧[1] 战文斌[1] 

机构地区：[1]中国海洋大学水产学院免疫与病害学实验室,山东青岛266003

出　　处：《中国海洋大学学报（自然科学版）》2017年第9期46-54,共9页Periodical of Ocean University of China

基　　金：山东省科技发展计划项目(2014GNC111015);山东省自主创新成果转化项目(2014ZZCX06205);国家自然科学基金项目(31472295;31672685)资助~~

摘　　要：本文将抗原芯片法原理与竞争免疫层析技术相结合,利用兔抗溶藻弧菌(Vibrio alginolyticus)多克隆抗体标记20nm胶体金颗粒后制得金标垫,将溶藻弧菌的外膜蛋白、鞭毛蛋白、胞外产物和全菌破碎蛋白4种抗原组分以及羊抗兔IgG喷涂在硝酸纤维素膜上,分别作为4条检测线和1条质控线,制备溶藻弧菌胶体金免疫层析快速检测试纸,并确定检测结果的判定方法。使用该试纸同时对溶藻弧菌、副溶血弧菌(V.parahaemolyticus)、哈维氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)和鱼肠弧菌(V.ichthyoenteri)进行检测,检查结果显示其能够准确区分溶藻弧菌感染与其他弧菌的交叉反应,排除了交叉反应对结果判定的干扰。溶藻弧菌快速检测试纸的最低检测限为5×10~5cfu·mL^(-1),检测时间为10~15min。该试纸具有操作简便、快速、准确、特异性强的优点,适用于普通人员在养殖现场对鱼类溶藻弧菌的快速检测。Anti-Vibrio alginolyticuspolyclonal antibody was labeled with freshly prepared colloidal gold particles with a 20 nm diameter. Four specific antigens of V. alginolyticus, including outer membrane protein, flagellum protein, extracellular products and whole-cell bacterial protein, and goat anti-rabbit IgG were dispensed on the nitrocellulose membrane as four test lines and the control line, respectively, and the colloidal gold test strip for rapiddetection of V. alginolyticus was developed through competitive immuno- chromatography combined with the antigen microarray principle. The developed colloidal gold test strip could distinguish the infection of V. alginolyticus from V. parahaemolyticus, V. harveyi, V. anguilla- rumand V. ichthyoenteri, and accurately tell the difference between V. alginolyticus infection and cross- reactivity of other bacteria. The test strip gave a detection limit of 5 × 10^5 cfu ·mL-1 of V. alginolyticus, and the testing took 10;15 min and presented a visualizing result. This easy-to-use test strip allowed onsite rapid detection of V. alginolyticus without the requirement of specialized equipment and professional personnel, providing a potential application prospect in fish aquaculture.

关 键 词：溶藻弧菌 胶体金 竞争免疫层析法 特异性抗原 免疫交叉反应 抗原芯片原理 快速检测试纸 

分 类 号：S94[农业科学—水产养殖]