鱼肠道弧菌间接ELISA快速检测法的建立

Development of Indirect ELISA for Rapid Detection of Bacterium Vibrio ichthyoenteri

为准确、快速诊断鱼肠道弧菌感染所致的海水鱼类全身性败血症,以鱼肠道弧菌为抗原,制备抗鱼肠道弧菌多克隆抗体,建立了鱼肠道弧菌间接酶联免疫吸附检测方法。该方法最佳反应条件为:抗原包被密度为107 cfu/mL,免疫血清稀释度为1∶211。病原菌检测灵敏度为每孔105 cfu/mL。抗血清与迟缓爱德华氏菌、杀鲑气单胞菌、恶臭假单胞菌、鱼肠道弧菌、副溶血弧菌、鳗弧菌、溶藻胶弧菌、秦皇岛弧菌8种水产致病菌的交叉反应结果呈阴性。用该法检测人工感染后的牙鲆和健康牙鲆,在发病鱼的肝脏、肾脏、脾脏、腹水等处检测出病原菌而健康鱼则为阴性。该技术能够准确、快速检测出鱼肠道弧菌。

In this article, indirect Enzyme Linked Immunosorbent Assay （ELISA） was established for rapid diagnosis of pathogenic bacterium Vibrio ichthyoenteri, a pathogen of whole body septicemia, which caused heavy loss in marine fish culture. Antiserum specific for the pathogenic bacterium was prepared from a New Zealand white rabbit and used in indirect ELISA. The optimal rapid diagnosis of the pathogenic bacterium was found using indirect ELISA under the conditions of the working antigen concentration of 107 cfu/mL, and working concentration of rabbit IGg purified from polyclonal antibody and diluted with distilled water of 1 ： 211. The minimal detectable concentration of the pathogenic bacterium was found to be 10s cfu/mL. Cross-reactions of antisera with other bacteria proved that the method was specificity, and had no cross-reaction with Vibrio parahaemoIyticus, V. alginolyticus, V. qinhuangdaora, V. anguillarum, Edwardsiella tarda, Aeromonas salmonicida, Pseudomonas putida and Escherichia coll. The ELISA method was applied to detect the strains isolated from artificial infected and healthy Japanese flounder （Paralichthys olivaceus）. The positive reaction was observed in the challenged Japanese flounder, that is, the V. ichthyoenteri was detected in liver, spleen, kidney and gill. However, the negative reaction was found in the healthy Japanese flounder, which no V. ichthyoenteri was detected, indicating that the indirect ELISA will facilitate the development of rapid diagnostic techniques for V. ichthyoenteri.