适配体捕获磁珠富集大肠埃希菌O157:H7方法的建立

Enrichment of E.coli O157:H7using aptamer capture magnetic beads

目的建立一种低浓度大体积样品中快速捕获富集大肠埃希菌O157:H7的方法。方法利用链霉亲和素-生物素结合特性,将生物素标记大肠埃希菌O157:H7特异性适配体与链霉亲和素磁珠相结合,制备捕获磁珠,捕获样品中的大肠埃希菌O157:H7,通过平板培养计数法检测捕获效率。结果通过优化反应条件,建立的适配体捕获磁珠富集法在O157:H7低浓度样品(10^3cfu/ml)中捕获效率≥92.49%,大肠埃希菌O157:H7浓度10^1cfu/ml时捕获效率为96.08%,高浓度样品(10^7cfu/ml)中捕获效率≥56.52%,在模拟实际样品(牛奶、果汁、汽水)中捕获效率均≥70%,与PCR法结果一致。结论基于免疫磁珠技术建立的大肠埃希菌快速捕获富集方法,具有特异性强、灵敏度高操作简单、成本低廉等特点,在实际检测中具有良好发展前景。

Objective To establish a method for rapid capture and enrichment of E.coli O157:H7in low-concentration and large-volume samples.Methods Using the binding characteristics of streptavidin and biotin,a biotin-labeled E.coli O157:H7-specific aptamer was combined with streptavidin magnetic beads.The system was prepared to capture mag-netic beads under optimal conditions and to capture E.coli O157:H7in the sample,and capture efficiency was determined using the plate counting method.Results The optimal capture system has an aptamer concentration of 200nM and an incubation time of 90min.The method of aptamer capture and enrichment of a microbe on magnetic beads as was established in this study captured≥92.49%of E.coli O157:H7in samples with a low concentration(10^3 CFU/ml).The capture efficiency was 96.08%at a concentration of 10^1 CFU/ml of E.coli O157:H7.The capture efficiency in the sample(10^7 CFU/ml)was≥56.52%.The results of this method are consistent with those of PCR,and E.coli O157:H7 was detected in all simulated samples.The capture efficiency in the simulated samples(milk,juice,and soda)was≥70%.Conclusion The rapid capture and enrichment of E.coli O157:H7based on immunomagnetic bead technology is highly specific,sensitive,simple,and inexpensive,and this method has good prospects in use in actual testing.