摘要
应用本实验室分离的国内首株羊肠道病毒(CEV)-JL14VP1重组蛋白制备的兔源多克隆抗体和抗CEV-JL14病毒的单克隆抗体,建立了检测CEV抗原的双抗体夹心ELISA方法,并对吉林省和内蒙古地区羊群感染CEV进行了病原流行病学调查。方阵试验确定了CEV VP1鼠源IgG捕获抗体的最佳包被量为0.2μg/孔,酶标抗体的最佳稀释倍数为1∶1 000。对大量CEV阴性粪便样品进行检测与统计学处理,确定了双抗体夹心ELISA检测CEV的判定标准为样品D490值≥0.216判定为阳性。特异性、敏感性和重复性试验结果表明,所建立的检测CEV抗原方法具有特异、敏感、快速和重复性好等特点。以建立的双抗体夹心ELISA方法,对吉林省和内蒙古不同地区的羊群粪便样品进行了检测,发现不同地区的羊群都存在着严重的CEV感染,感染率高达12%~100%。本研究为今后CEV感染的诊断、检疫及防制提供了有效的手段和流行病学理论依据。
Enterovirus infection in sheep/goat is an emerging infectious disease worldwide, which poses a severe threat to the development of sheep/goat industry. In this study,we reported the de- velopment of a double antibody sandwich ELISA for detecting caprine/ovine enterovirus antigens using the antibodies generated against the recombinant proteins VP1 of caprine enterovirus JL14 (CEV-JL14) ,and investigated the CEV infections in sheep/goat herds in Jilin province and Inner Mongolia Automonous region. The concentrations of coating-antibody and HRP-conjugated anti- VP1 were optimized using square matrix titrimetry. The criterion for sandwich ELISA was deter- mined and sample was assessed as positive when its D490≥0. 216. The established ELISA had high sensitivity,specificity and simplicity. Epidemiological survey demonstrated that CEV infection in sheep/goat herds in Jilin province and Inner Mongolia Automonous region was up to 12%-100%. The findings in this study will provide a sensitive, specific, simple and rapid antigen detection method for diagnosis and epidemiologic survey of CEV infection,and an invaluable data for preven tion and control of this emerging infectious diseases.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2017年第4期653-657,共5页
Chinese Journal of Veterinary Science
基金
"十三五"国家重点研发计划"畜禽重大疫病防控与高效安全养殖综合技术研发"重点专项"牛羊重要疫病诊断与检测新技术研究"资助项目(2016YFD0500904)
国家自然科学基金资助项目(31572531)