牦牛肠道病毒间接免疫荧光检测方法的建立与初步应用  

Development and Preliminary Application of an Indirect Immunofluorescent Assay of the Yak Enterovirus

作　　者：何欢[1] 叶勇刚[2] 陈新诺 王斌星[1] 覃思楠 刘燕[1] 汤承[1,3] 张斌[1,3] 

机构地区：[1]西南民族大学生命科学与技术学院,成都610041 [2]四川省畜牧科学研究院,成都610066 [3]国家民委青藏高原动物疫病防控创新团队,成都610041

出　　处：《畜牧兽医学报》2016年第12期2450-2456,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金："十三五"国家重点研发计划(2016YFD0500907);四川省科技计划项目青年基金(2014JQ0044);四川省教育厅创新团队项目(13TD00527);四川奶牛传染系流产的病原调查研究(SASA2015A12);西南民族大学中央高校基本科研业务费专项资金项目(2016NGJPY10)

摘　　要：为了建立准确、快速的牦牛肠道病毒(yak enterovirus)检测方法,利用本实验室已获得的一株牦牛肠道病毒SWUN-AB001,免疫健康兔获得抗SWUN-AB001高免血清,建立了一种检测牦牛肠道病毒的间接免疫荧光试验(IFA)方法,并对其工作条件和时间进行筛选,优化出最佳条件。采用该方法对40份临床腹泻样本进行了牦牛肠道病毒的检测,并与RT-PCR的检测结果进行比较。结果表明:获得的高免血清在该IFA方法中的最佳稀释度为1∶100,工作时间为30min;FITC标记的羊抗兔IgG(二抗)的稀释度为1∶800,工作时间为45min,牦牛肠道病毒特异性亮绿色荧光颗粒清晰可见;同时与牛病毒性腹泻病毒、轮状病毒、大肠杆菌、沙门菌、葡萄球菌和空肠弯曲杆菌反应均未见明显的荧光颗粒;该方法能够检测SWUN-AB001病毒的最低浓度约为20TCID50·mL-1;该方法的临床检测结果与RT-PCR的检测结果之间无差异(P=0.329)。结果表明该方法特异性强、重复性好、灵敏度高,为该病毒在牦牛养殖地区的流行病学调查、临床诊断和快速诊断试剂的开发奠定了基础。To establish an method for rapid and accurate detection of the yak enterovirus,the yak enterovirus SWUN-AB001 obtained by our laboratory was used to immunize healthy rabbits,and then establish an indirect immunofluorescent assay（IFA）by using the rabbit hyperimmune serum.And the working conditions and time were screened and optimized.The best dilution and working time of hyperimmune serum in the IFA method were 1：100and 30 minutes,and these of FITC conjugated goat anti-rabbit IgG were 1：800and 45 minutes.Specificity of bright green fluorescent particles about the yak enterovirus were clearly visible;it can′t react with Bovine viral di-arrhea virus,Rotavirus,E.coli,Salmonella,Streptococcus,Staphylococcus and Pasteurella;the minimum concentration of SWUN-AB001 can be detected by the method was about20TCID50·mL-1.Forty clinical diarrhea samples were detected by using the RT-PCR and the IFA,and there was no difference in the experiment results between the RT-PCR and the IFA（P=0.329）.The results indicate that the method has strong specificity,good repeatability and high sensitivity,it can be used in study of epidemiological investigation,clinical diagnosis and laid a foundation of the fast diagnostic reagents.

关 键 词：牦牛 肠道病毒 间接免疫荧光检测方法 

分 类 号：S852.659.6[农业科学—基础兽医学]