肠出血性大肠杆菌O157胶体金免疫层析试纸条的研制

目的建立一种简便实用的胶体金免疫层析检测方法用于食品样品中肠出血性大肠杆菌O157的快速筛查。方法采用柠檬酸三钠还原法制备胶体金,以抗EHEC O157 LPS单克隆抗体标记,以兔抗O157多克隆抗体固定于硝酸纤维素膜作为捕获抗体,利用双抗夹心法,制成O157胶体金免疫层析检测试纸条,并进行了特异性和敏感性试验。结果该试纸条仅与O157反应,与其他受试菌株均不反应。检测敏感性为106CFU/mL,对人工污染EHEC O157模拟牛肉样品增菌后检测,敏感性为56 CFU/g。结论成功研制出EHEC O157胶体金免疫层析试纸条,敏感性高,特异性强,检测速度快,10min可出结果,操作简便,可用于现场大量样品的检测。

Development of immunochromatographic strip for detection of enterohemorrhagic Escherichia coli O157

To develope immunochromatographic(IC) strip for the rapid detection of enterohemorrhagic Escherichia coli(EHEC) O157 in foof samples,murine monoclonal antibody 3D6 to EHEC O157 lipopolysaccharide was conjugated with colloidal gold particles prepared by the citrate method,and the polyclonal antibody was immobilized on nitrocel lulose membrane to prepare an IC strip device.The specificity of the IC strip was determined using 21 pure-cultured bacteria,including 10 E.coli strains and 11 non-E.coli strains.It was found that all the three EHEC O157 strains produced a positive signal,whereas the others did not.The sensitivity of the IC strip was determined using 10-fold diluted EHEC O157∶H7,with a range of 5.6×107 to 56 CFU g-1 in raw beef after enrichment.These results indicated that the IC strip exhibited high specificity and sensitivity to detect EHEC O157,and it is assay is rapid,economical,and simple,without requirement of complicated equipment.