鱼肠道弧菌胶体金快速检测试纸研制

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...

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

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 the detection time was 10 min. To ensure the accuracy of the detection, tissues of flounder (Paralichthys olivaceus) infected by V. ichthyoenteri were detected using the test strip and ELISA simultaneously, and positive results were obtained with both methods. This study provides a promising on-site tool for rapid detection of V. ichthyoenteri in fish aquaculture.