非洲猪瘟病毒p30和pK205R双抗原间接ELISA检测方法的建立  

Establishment of indirect ELISA based on p30 and pK205R dual-proteins for detection of African swine fever virus

作　　者：何佳依 邹艳丽[2] 王燕 刘珊[2] 苗雨润 任炜杰[2] 王振忠 白昀[3] 陈欢 贾红[4] 郑龙三 吴晓东[2] 冯志新[3] 钱莺娟[1] HE Jiayi;ZOU Yanli;WANG Yan;LIU Shan;MIAO Yurun;REN Weijie;WANG Zhenzhong;BAI Yun;CHEN Huan;JIA Hong;JUNG Yongsam;WU Xiaodong;FENG Zhixin;QIAN Yingjuan(Key Laboratory of Animal Bacteriology,Ministry of Agriculture/MOE Joint International Research Laboratory of Animal Health and Food Safety/College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;National African Swine Fever Reference Laboratory/China Animal Health and Epidemiology Center,Qingdao 266032,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]南京农业大学动物医学院教育部动物健康与食品安全国际合作联合实验室农业部动物细菌学重点实验室,江苏南京210095 [2]中国动物卫生与流行病学中心国家非洲猪瘟参考实验室,山东青岛266032 [3]江苏省农业科学院兽医研究所,江苏南京210014 [4]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国兽医科学》2024年第11期1443-1450,共8页Chinese Veterinary Science

基　　金：“十四五”国家重点研发计划项目(2021YFD1801200,2022YFD1800500);国家重点研发计划政府间国际科技创新合作重点专项(2019YFE0107300);江苏省重点研发计划(现代农业)重点项目(SBE2020310346)。

摘　　要：为建立一种可靠、快速的血清学检测方法,以非洲猪瘟病毒(ASFV)结构蛋白p30和非结构蛋白pK205R作为包被抗原,建立了一种双抗原间接ELISA方法,用于检测ASFV抗体。结果表明,该方法与伪狂犬病毒、猪繁殖与呼吸综合征病毒、猪肺炎支原体、猪圆环病毒和猪瘟病毒等常见猪源病原阳性血清无交叉反应;对ASFV阳性血清的敏感性能达到1∶12800;批内变异系数为7.95%,批间变异系数为9.18%,均小于10%。利用该方法与ID.vet商品化ELISA试剂盒分别检测130份临床猪血清,其阳性符合率为92.19%,阴性符合率为86.36%,总符合率为89.23%。综上所述,成功建立了ASFV p30和pK205R双抗原间接ELISA检测方法,为检测猪血清中ASFV特异性抗体提供了新的技术支持。Key Laboratory of Animal Bacteriology,Ministry of Agriculture/MOE Joint International Research Laboratory of Animal Health and Food Safety/College of Veterinary Medicine,Nanjing Agricultural University;National African Swine Fever Reference Laboratory/China Animal Health and Epidemiology Center;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences;In order to develop a reliable and rapid serological detection method,a dual-antigens indirect ELISA was established with structural protein p30 and non-structural protein pK205R of African swine fever virus(ASFV)as diagnostic antigens for the detection of ASFV antibodies.The results showed that the method had no cross-reactivity with positive sera of common swine-origin pathogens,such as pseudorabies virus,classical swine fever virus,Mycoplasma hyopneumoniae,porcine circovirus and porcine reproductive and respiratory syndrome virus.The sensitivity to ASF positive serum reached1∶12800.The coefficient of variation of intra-batch was 7.95%and the coefficient of variation of inter-batch repeatability was 9.18%,both less than 10%.Total 130 clinical pig serum samples were tested by the method and a ID.vet commercial ELISA kit,the positive conformity rate reached 92.19%,the negative conformity rate reached 86.36%,and the total conformity rate reached 89.23%.In summary,this study successfully established an ASFV p30 and pK205R dual-antigens indirect ELISA,which provided a new technical support for the detection of ASFV-specific antibodies in pig serum.

关 键 词：非洲猪瘟病毒 p30蛋白 pK205R蛋白 间接ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]