鱼肠道弧菌胶体金快速检测试纸研制  

Development of a competitive gold immunochromatography test strip for rapid detection of Vibrio ichthyoenteri in fish

作　　者：绳秀珍[1] 王新趁 李嘉文 唐小千[1] 邢婧[1] 迟恒[1] 战文斌[1,2] SHENG Xiuzhen;WANG Xinchen;LI Jiawen;TANG Xiaoqian;XING Jing;CHI Heng;ZHAN Wenbin(Laboratory of Pathology and Immunology of Aquatic Animals,KLMME,Ocean University of China,Qingdao 266003,China;Laboratory for Marine Fisheries Science and Food Production Processes,National Laboratory for Marine Science and Technology,Qingdao 266071,China)

机构地区：[1]中国海洋大学水产动物病害与免疫学实验室,KLMME,山东青岛266003 [2]青岛海洋科学与技术国家实验室,海洋渔业科学与食物产出过程功能实验室,山东青岛266071

出　　处：《中国水产科学》2021年第1期1-11,共11页Journal of Fishery Sciences of China

基　　金：国家重点研发计划项目(2019YFD0900101);国家自然科学基金项目(31872599,31730101,31672685);山东省泰山学者特聘专家项目.

摘　　要：鱼肠道弧菌(Vibrio ichthyoenteri)可引起多种养殖鱼类发病死亡,给鱼类养殖业带来严重经济损失。为解决养殖过程中鱼肠道弧菌的现场快速检测问题,本研究研制了鱼肠道弧菌胶体金快速检测试纸。通过制备兔抗鱼肠道弧菌多克隆抗体,间接ELISA分析发现其与鱼肠道弧菌的外膜蛋白、鞭毛蛋白、胞外产物及全菌破碎蛋白发生阳性免疫反应,与副溶血弧菌(Vibrio parahaemolyticus)、鳗弧菌(Vibrio anguillarum)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)的4种抗原蛋白发生程度不等的免疫交叉反应。制备胶体金标记鱼肠道弧菌抗体,确定了鱼肠道弧菌4种抗原蛋白的划线浓度,以4种抗原蛋白作为4条检测线,羊抗兔IgG作为质控线,基于竞争法免疫层析技术研制出鱼肠道弧菌快速检测试纸。对鱼肠道弧菌、副溶血弧菌、鳗弧菌、溶藻弧菌和哈维氏弧菌的检测结果显示,该试纸可以准确鉴别出鱼肠道弧菌,并能判别其他病原菌的交叉反应。该试纸对鱼肠道弧菌的最低检测限为5×10^5 CFU/mL,检测耗时为10 min。对患病牙鲆组织的检测结果显示,该试纸与ELISA结果一致,表明具有较好的检测准确性。本研究为水产养殖现场的鱼肠道弧菌快速、准确检测提供了有力工具。Vibrio ichthyoenteri is one of the most common pathogenic bacteria in marine-cultured fish that can infect many fish species,causing serious economic losses in fish aquaculture.Rapid diagnostic testing is critical for disease prevention and control.This study was performed in order to produce a method to detect V.ichthyoenteri on-site easily,rapidly,and accurately.First,rabbit anti-V.ichthyoenteri antibody was prepared,and the outer membrane protein,flagellum protein,extracellular products,and whole-cell bacterial protein were extracted from V.ichthyoenteri,Vibrio parahaemolyticus,Vibrio anguillarum,Vibrio alginolyticus,and Vibrio harveyi.The immunoreaction between the anti-V.ichthyoenteri antibody and the antigen proteins were subsequently analyzed;the results showed a strong positive immunoreaction between the anti-V.ichthyoenteri antibody and the four antigens of V.ichthyoenteri.However,cross-immunoreaction was observed between the anti-V.ichthyoenteri antibody and antigens of other vibrios.Thereafter,the anti-V.ichthyoenteri antibody was labeled with colloidal gold nanoparticles,and the concentration of the four specific antigens of V.ichthyoenteri for preparation of test lines were confirmed by ELISA.To avoid the interference of cross-reactivity,the four specific antigens of V.ichthyoenteri were dispensed on nitrocellulose membrane at different concentrations as four test lines(T1-T4);the whole-cell bacterial protein was set on the line T4,and goat-anti-rabbit IgG was used as the control line.A test strip for rapid detection of V.ichthyoenteri was thus developed based on competitive immunochromatography.The presence of V.ichthyoenteri,V.parahaemolyticus,V.anguillarum,V.alginolyticus,and V.harveyi could be detected with this test strip,and the results showed that it could specifically distinguish V.ichthyoenteri and exclude the interference of cross-reactivity of the other four bacteria,indicating good specificity.The detection limit of the test strip was determined to be 5×105 CFU/mL for V.ichthyoenteri,and

关 键 词：鱼肠道弧菌 胶体金 检测试纸 竞争免疫层析法 免疫交叉反应 

分 类 号：S941[农业科学—水产养殖]