双抗体夹心ELISA检测小反刍兽疫病毒抗原方法的建立及病原流行病学调查  

Development of a direct sandwich ELISA for PPRV antigen detection and its application in PPRV epidemiological investigation

作　　者：郭昌明[1] 张群[1] 刘亚静[1] 鲁海冰 邢泽黎 邓颖瑞 朱利塞[1] 郭淳光 王新平[1,2] 

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]吉林大学教育部人兽共患病研究所,吉林长春130062

出　　处：《中国兽医学报》2017年第5期799-803,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"基金资助项目(2016YFD0500900)

摘　　要：应用表达纯化的小反刍兽疫病毒(peste des petits ruminants virus,PPRV)N蛋白制备的多克隆抗体,建立了检测PPRV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PPRV进行了调查。方阵法确定了抗PPRV兔源IgG作为捕获抗体的包被量为0.2μg,酶标抗体的最佳稀释度为1∶1 000。对大量小反刍兽疫阴性粪便样品进行检测及统计学处理,确定了双抗体夹心ELISA检测PPRV的判定标准,即被检粪便样品D490≥0.221,判定为阳性。特异性、敏感性等试验结果表明,建立的检测PPRV抗原方法具有特异、敏感和快速等优点。与RT-PCR方法相比,该方法省时省力、简单快速。应用建立的检测PPRV抗原的双抗体夹心ELISA对吉林省和内蒙古不同地区的羊粪样进行检测,发现羊群均存在程度不同的PPRV隐性感染。本研究在国内首次揭示出临床健康羊群携带PPRV,为今后小反刍兽疫的诊断与防控提供了新的流行病学理论依据。A sandwich ELISA for detection of peste des petits ruminant virus （PPRV） antigens was developed using antibodies generated against the GST-fusion N proteins derived from PPRV, and used to investigate the potential PPRV infections in sheep/goat herds in Jilin province and Inner Mongolia Autonomous region. The concentrations for antibody coating and antibody conjugated with HRP were optimized using square matrix titrimetry. The criterion for sandwich ELISA was expressed as the average of D490 plus 3 standard deviation from 48 PPRV-negative feces samples and determined as positive when sample＇s D490 ≥0. 221. The established sandwich ELISA was dem- onstrated to have a high specificity and sensitivity with a good repeatability. Detection of the fecal samples from Jilin province and Inner Mongolia autonomous regions using the established ELISA revealed a various subclinical infection in ,clinically healthy sheep/goat herds, which was further confirmed by the electron microscopy and RT-PCR. The findings in this study provide a solid evidence for the PPRV subclinical infections challenge for the prevention and control of among sheep/goats herd at home, thus providing new this emerging disease in China.

关 键 词：PPRV 小反刍兽疫 双抗体夹心ELISA 隐性感染 流行病学调查 

分 类 号：S852.65[农业科学—基础兽医学]