蓝狐兔脑炎微孢子虫夹心ELISA检测方法的建立与应用  

Development and availability of a sandwich ELISA for the quantification of Encephalitozoon cuniculi in the blue fox(Alopex lagopus)

作　　者：周纪元 穆国冬[2] 于鹏 刘爱鑫 马哲昊 张博[1] 周井祥[1] 王好[1] 刘丽凡 ZHOU Ji-yuan;MU Guo-dong;YU Peng;LIU Ai-xin;MA Zhe-hao;ZHANG Bo;ZHOU Jing-xiang;WANG Hao;LIU Li-fan(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Jilin Province Animal Disease Prevention and Control Center,Changchun 130000,China;Graduate School,Changchun University,Changchun 130022,China)

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林省动物疫病预防控制中心,吉林长春130000 [3]长春大学研究生院,吉林长春130022

出　　处：《中国兽医科学》2022年第9期1094-1101,共8页Chinese Veterinary Science

基　　金：吉林省科技厅科技支撑计划重点项目(20190301009NY)。

摘　　要：本试验旨在建立一种可用于大规模检测蓝狐兔脑炎微孢子虫的夹心ELISA方法,使其在疾病早期或隐性感染时,能及早地发现并确诊,从而采取措施。首先,于病料中初步分离兔脑炎微孢子虫,随后通过革兰染色和ITS基因对比验证病原,通过接种犬肾细胞进行纯化。制定免疫方案,将纯化的兔脑炎微孢子虫以1×10^(7)mL^(-1)免疫新西兰白兔制备多克隆抗体。采用纯化后的多抗作为捕捉抗体,蓝狐Ig G作为识别抗体,家兔抗狐Ig G-HRP作为指示抗体,通过检测纯化后的微孢子虫建立夹心ELISA。研究表明,多抗最适包被质量浓度为5μg/m L,50 g/L脱脂奶粉溶液37℃封闭1 h,待测样本孵育时间为2 h(37℃),识别抗体质量浓度为37.5μg/m L,反应时间为1.5 h(37℃),家兔抗狐Ig G-HRP工作浓度为1∶5000,孵育时间为1 h(37℃)。本研究建立的夹心ELISA对纯化孢子的最低检测浓度为6.25×10^(5)mL^(-1),对人工添加微孢子虫的最低检测灵敏度为1.25×10^(6)mL^(-1),特异性试验时只在检测添加兔脑炎微孢子虫时为阳性。通过检测辽宁省辽阳市不同养殖场的60份样品,对建立的夹心ELISA进行初步应用,其结果与PCR检测结果的符合率为79.97%。本研究具有一定的可应用性,可为大规模检测蓝狐感染兔脑炎微孢子虫时提供一些技术支持,同时也为兔脑炎微孢子虫的免疫学检测方法的研究提供了理论依据。This experiment aims to establish a sandwich ELISA that can be used for a large-scale detection for Encephalitozoon cuniculi,for detection and diagnosis in the early stage of the disease or latent infection,and further treatment.First,E.cuniculi was isolated from disease samples,and then verified by Gram staining and ITS gene comparison,and then were purified using the inoculating canine kidney cells.An immunization protocol was developed as immunize New Zealand white rabbits with purified E.cuniculi spores at 1×10^(7)mL^(-1)to prepare polyclonal antibodies,and then the purified polyclonal antibody was used as the capture antibody,blue fox Ig G as the recognize antibody,and rabbit anti-fox Ig G-HRP as the indicator antibody.Therefore,the sandwich ELISA was established by detecting the purified E.cuniculi.The research suggested that the optimal coating mass concentration of the polyclonal antibodies was 5μg/mL,the optimal blocking condition was 50 g/L nonfat milk powder solution at 37℃ for 1 h.The incubation condition for sample was at 37℃ for 2 h,the recognize antibody mass concentration was 37.5μg/mL at 37℃ for 1.5 h,the working concentration of rabbit anti-fox Ig G-HRP was 1∶5000 at 37℃ for 1 h.The minimum detection concentration of the sandwich ELISA for purified E.cuniculi was 6.25×10^(5)mL^(-1),and that for the artificially added microsporidia was 1.25×10^(6)mL^(-1).In the specificity test,only the addition of E.cuniculi was detected to be positive.By detecting 60 samples from different farms in Liaoyang City,Liaoning Province,we preliminarily applied the sandwich ELISA,and the compliance ratio between the ELISA detection result and PCR detection result was 79.97%.This research possess certain applicability,as a technical support for the large-scale detection of E.cuniculi.Meanwhile,this research provides a theoretical basis for studying immunological detection method of E.cuniculi.

关 键 词：蓝狐 兔脑炎微孢子虫 多克隆抗体 夹心ELISA 

分 类 号：S852.7233[农业科学—基础兽医学]