新型凝集技术快速检测产肠毒素性大肠杆菌菌毛的研究

Research of new agglutination test for rapid detection of ETEC fimbriae

为建立以彩色二氧化硅微球(CSN)为载体,快速检测产肠毒素性大肠杆菌(ETEC)菌毛K88ab、K99、987p和F41的新型凝集技术,本研究采用反相微乳液法制备二氧化硅微球,将其与菌毛单因子血清偶联制成免疫彩色二氧化硅微球(ICSN),通过优化反应条件建立了快速检测ETEC菌毛的方法。特异性试验结果显示,ICSN只与携带其对应菌毛的菌株发生反应,与携带其它菌毛的菌株无交叉反应;敏感性试验结果显示菌液的最小检出浓度为1.0×10~6cfu/m L^5.1×10~6cfu/m L;稳定性试验显示4℃保存30 d,ICSN特异性、敏感性无明显变化;双盲样品检测准确率为100%。本研究有助于ETEC菌毛的快速检测和幼畜大肠杆菌性腹泻的诊断。

To establish a new agglutination technique for detection of Enterotoxigenic Escherichia coli （ETEC） pili K88ab, K99, 987P and F41 genes based on colored silica microspheres （CSN） synthesized by reverse microemulsion method. The CSN surface was modificated by fimbriae mono-specific serum to prepare immune colored silica microspheres （ICSN）. Then the method for rapid detection of ETEC pilus was established by optimization of reaction conditions. The results showed that the prepared ICSN was only reacted with the corresponding strains and there was no cross reaction with the other strains, and the detection limit was 1.0×106 cfu/mL-5.1×106 cfu/mL. The specificity and sensitivity of ICSN preserved at 4 ℃ for 30 days had no obvious change. The accuracy rate of the detection for double blind samples was 100%. This research established an effective method for detection of ETEC fimbriae and the diagnosis of piglets and calves diarrhea caused by ETEC.