创伤弧菌快速检测试纸条的研制

Development of an immunochromatographic strip test for rapid detection of Vibrio vulnificus

目的研制一种敏感、特异、快速的免疫试纸条,用于创伤弧菌检测。方法用创伤弧菌（ATCC27562）菌体抗原免疫家兔,制备兔抗创伤弧菌多克隆抗体;采用柠檬酸盐还原法制备胶体金颗粒并标记多克隆抗体,纯化后滴加在玻璃纤维素膜上;在硝酸纤维素膜上包被兔抗创伤弧菌多克隆抗体和羊抗兔IgG,分别作为检测线和质控线;将处理后的硝酸纤维膜、玻璃纤维素膜与样品垫、吸水纸组装成试纸条,4℃保存并定期检测;设阳性及阴性对照,对本试纸条检测的灵敏度、特异性进行检测。结果将试纸条插入样品溶液中,20~30min内即可显示结果,其检测灵敏度为2×106CFU/ml;特异性检测显示本试纸条与溶血性葡萄球菌、金黄色葡萄球菌、副溶血弧菌等12种菌株均无交叉反应;稳定性检测表明该试纸条可4℃保存4个月。结论本研究构建出创伤弧菌多克隆检测试纸条,具有较高的灵敏度和较好的特异性,简便,20~30min即可获得检测结果,可用于创伤弧菌的快速检测。

Objective To establish a rapid strip test for detection of Vibrio vulnificus. Method Anti-Vibrio vulnificus polyclonal antibodies were obtained from the rabbits immunized with Vibrio vulnificus（ATCC27562） . Colloidal gold was prepared through reducing HAuCl4·4H2O by sodium citrate and conjugated with the polyclonal antibodies as the detecting reagent. The polyclonal antibodies and sheep anti-rabbit immunoglobulins were separately coated onto the same nitrocellulose membrane for sample detection and quality-control,respectively. The nitrocellulose membrane,gold conjugate pad,sample pad,filter paper and absorbent pad were assembled to prepare the strips. The detection specificity and sensitivity of this strip were evaluated. Results The strip test for detecting Vibrio vulnificus yielded results in 20 to 30 min. The detection sensitivity of the test was 2×106 CFU/ml. The strip showed no cross-reaction with other bacterial strains. The strips remained stable after preservation at 4 ℃ for 4 months. Conclusion With a high specificity and sensitivity,this strip test is applicable in the detection of Vibrio vulnificus.