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

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.