嗜水气单胞菌胶体金快速检测试纸条的研制

用柠檬酸三钠还原氯金酸制备胶体金颗粒,标记抗嗜水气单胞菌单克隆抗体并制备金标垫。将吸收垫、喷涂有抗嗜水气单胞菌单克隆抗体和羊抗鼠抗体的硝酸纤维素膜、金标垫及样品垫组装成免疫层析试纸条,建立嗜水气单胞菌的胶体金免疫层析快速检测方法。用灭活细菌与血清混合的模拟检测样本测定试纸条的特异性、灵敏度,结果显示:试纸条对豚鼠气单胞菌、温和气单胞菌、鳗弧菌、溶藻弧菌、荧光假单胞菌、副溶血弧菌等13种常见病原菌没有交叉反应,与嗜水气单胞菌有特异性反应,检测灵敏度为1×105CFU/mL,检测时间小于5min。所制备的嗜水气单胞菌胶体金免疫层析检测试纸条具有快速、简便、特异性高、适用于基层临床生产推广应用等优点。

Development of a rapid colloidal gold immunochromatographic strip test for the detection of Aeromonas hydrophila

For the aim of rapid and simple examination of Aeromonas hydrophila,gold immunochromatographic lateral flow assay system(GICA) was developed.In the GICA test strip system,monoclonal antibody(McAb) 1F3 against A.hydrophila was used as a capture antibody at the test line,goat anti-mouse IgG antibody was used as a capture antibody at the control line on the nitrocellose membrane.McAb 1A5 conjugated with colloidal gold acts as signal generator on conjugate pad which attached to the nitrocellose membrane.The preliminary feasibility study of this method was as follows:the sensitivity of the GICA test strip towards A.hydrophila was high with a detection limit of 1×105 CFU/mL,the specificity analysis showed no cross-reaction with other thirteen bacteria of aquaculture pathogens such as A.cavia,A.sobria,Vibrio anguillarum,V.alginolyticus,V.parahaemolyticus,Pseudomonas pseudomallei,et al.Accurate reading time for confirmation of the assay could be completed in 5 min with a liquid sample of 100 μL.The GICA test strip assay system constructed in the present study proved to be high sensitive and specific on the detection of A.hydrophila.The examination system was rapid,simple,inexpensive,and did not require any sophisticated equipment.