副溶血弧菌胶体金快速检测试纸的研制及应用  

Development of a Competitive Gold Immunochromatographic Test Strip for Rapid Detection of Vibrio parahaemolyticus in Fish

作　　者：朱慧 李嘉文 绳秀珍[1] 唐小千[1] 邢婧[1] 战文斌[1,2] ZHU Hui;LI Jia-Wen;SHENG Xiu-Zhen;TANG Xiao-Qian;XING Jing;ZHAN Wen-Bin(Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, Qingdao 266003, China;Function Laboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, 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,山东青岛266003 [2]青岛海洋科学与技术试点国家实验室,海洋渔业科学与食物产出过程功能实验室,山东青岛266237

出　　处：《中国海洋大学学报（自然科学版）》2021年第3期24-33,共10页Periodical of Ocean University of China

基　　金：国家重点研究发展计划项目(2019YFD0900101,2019YFD0900102);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资助。

摘　　要：副溶血弧菌(Vibrio parahaemolyticus)是海水养殖动物弧菌病的主要病原菌之一。为实现对该菌的现场快速检测,本文以制备的兔抗副溶血弧菌多克隆抗体作为金标抗体,提取副溶血弧菌的外膜蛋白、鞭毛蛋白、胞外产物和全菌蛋白,以不同浓度的4种抗原蛋白作为4条检测线(T1~T4),特别在T4线设置全菌蛋白,基于竞争免疫层析原理研制出副溶血弧菌胶体金快速检测试纸。使用该试纸检测了副溶血弧菌(V.parahaemolyticus)、鳗弧菌(V.anguillarum)、溶藻弧菌(V.alginolyticus)、哈维氏弧菌(V.harveyi)和鱼肠道弧菌(V.ichthyoenteri),检测结果表明其能够准确鉴别副溶血弧菌感染,排除其他菌的交叉反应干扰。副溶血弧菌快速检测试纸的最低检测限为5×105 cfu·mL^-1,感染副溶血弧菌的牙鲆组织的检测结果与ELISA结果一致。本文研制的试纸具有较高的特异性与准确性且操作简单,为现场快速检测副溶血弧菌提供了有力工具。Vibrio parahaemolyticus is one of the main pathogens of vibriosis in mariculture animals.In order to realize the rapid on-site detection of V.parahaemolyticus,this study was carried out.The rabbit polyclonal antibody against V.parahaemolyticus was produced and labeled with freshly prepared colloidal gold nanoparticles(20 nm in diameter).On nitrocellulose membrane,four specific antigen proteins of V.parahaemolyticus including outer membrane protein,flagellum protein,extracellular products and whole-cell bacterial protein were dispensed as four test lines,goat anti-rabbit IgG were dispensed as the control line,and a competitive colloidal gold immunochromatographic test strip for rapid detection of V.parahaemolyticus was developed.The developed colloidal gold test strip can distinguish the infection of V.parahaemolyticus from V.anguillarum,V.alginolyticus,V.ichthyoenteri and V.harveyi,and exclude the interference of cross-reactivity of other bacteria.The test strip gave a detection limit of 5×105 cfu·mL^-1 of V.parahaemolyticus.The tissues from V.parahaemolyticus-infected flounder were detected using the test strip and ELISA,and positive results were obtained with the two methods.This easy-to-use test strip provided a potential application in fish aquaculture.

关 键 词：副溶血弧菌 胶体金 竞争免疫层析法 抗原 免疫交叉反应 快速检测试纸 

分 类 号：S94[农业科学—水产养殖]