5种重要虫媒病毒液相蛋白芯片多重检测方法的建立及应用

Establishment and application of a method with liquid protein chip for multi-detection of 5 arboviruses

目的建立森林脑炎、乙型脑炎、登革热、基孔肯雅和刚果-克里米亚出血热5种虫媒病毒的液相蛋白芯片检测方法,为虫媒病毒的筛查检测提供一种新方法。方法将病毒抗原包被微球,采用间接法原理,利用病毒抗体和阳性血清建立并优化5种虫媒病毒液相蛋白芯片检测方法。对临床样本进行检测,评价液相蛋白芯片方法的敏感性、准确性。结果以病毒抗体和阳性血清建立的5种虫媒病毒液相蛋白芯片检测方法,病毒抗体及生物素标记二抗的孵育时间均为1 h,生物素标记羊抗兔/鼠/人Ig G的最佳稀释度为1∶100,能对5种虫媒病毒进行准确检测。该方法具有高通量、快速、敏感、特异等特点。结论本研究所建立的5种虫媒病毒液相蛋白芯片检测方法,可用于这5种虫媒病毒的同时筛查检测,适用于国境口岸对虫媒病毒进行筛查检测,能提高检测的效率,满足口岸检疫中快速检验的要求。

Objective To establish a method with liquid protein chip for the detection of 5 species of arboviruses simultaneously,including forest encephalitis virus,Japanese encephalitis virus,dengue fever virus,Chikungunya virus and Congo- crimean hemorrhagic fever virus,so as to provide a new method for the screening of arboviruses. Methods The antigens of arbovirus were coupled to different microspheres. With indirect method,the liquid protein chip method for the detection of 5 arbovirus separately was established and optimized by using virus antibody and positive serum. The clinical sample was detected and the sensitivity and accuracy of the method were analyzed. Results The method established using virus antibodies and positive serum for detection of 5 arbovirus with liquidchip was as follows,the incubation time of antibody and biotin- labeled secondary antibody was 1 h,the best dilution of biotinylated goat anti- rabbit / mouse / human Ig G was 1∶100. Detecting results show that the method has a feature of high- throughput,rapidity,sensitivity and specification in the detection of 5 arbovirus. Conclusion The method established for the detection of 5 arbovirus with liquid chip can be used for screening the 5 arbovirus at the same time,which is suitable for the screening and detection of arbovirus in frontier ports,can improve the detection efficiency,and meet the requirements for quick inspection in ports.