鉴别ASFV野毒株与CD2v缺失株抗体检测方法的建立及应用  

A diagnostic method for distinguishing wild ASFV strain from CD2v deletion strain

作　　者：王新凯 杨芷翊 朱忠武 林彦星 黄超华 王友令 曹琛福 贾伟新[1] WANG Xin-kai;YANG Zhi-yi;ZHU Zhong-wu;LIN Yan-xing;HUANG Chao-hua;WANG You-ling;CAO Chen-fu;JIA Wei-xin(African Swine Fever Regional Laboratory of China(Guangzhou),National Local Joint Engineering Laboratory of Zoonosis Prevention and Control Agents,Key Laboratory of Zoonoses of Ministry of Agriculture and Rural Affairs,Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture and Rural Affairs,Guangdong Key Laboratory forPrevention and Control of Zoonotic Diseases,College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China;Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center,Shenzhen 518045,China;Changhai Customs Technology Center,Changsha 410004,China;Guangzhou Danon Biotechnology Co Ltd,Guangzhou 510665,China)

机构地区：[1]华南农业大学,兽医学院,国家非洲猪瘟区域实验室(广州),人兽共患病防控制剂国家地方联合工程实验室,农业农村部人畜共患病重点实验室,农业农村部兽用疫苗创制重点实验室,广东省动物源性人兽共患病防控重点实验室,广东广州510642 [2]深圳海关动植物检验检疫技术中心,广东深圳518045 [3]长海海关技术中心,湖南长沙410004 [4]广州达农生物科技有限公司,广东广州510665

出　　处：《中国兽医科学》2023年第5期545-551,共7页Chinese Veterinary Science

基　　金：广东省科技计划项目(2021B1212030015);广东省重点领域研发计划项目(2019B2021003)。

摘　　要：本研究通过建立两种不同包被抗原的间接ELISA方法,旨在对ASFV野毒株与CD2v缺失株进行鉴别诊断。通过优化后的EP402R基因序列进行原核表达重组蛋白,纯化后作为包被抗原建立基于CD2v重组蛋白的间接ELISA方法;参考WOAH方法对灭活后的ASFV进行纯化,并作为包被抗原建立基于全病毒蛋白的间接ELISA方法。通过对抗原包被浓度等条件的优化,建立的基于CD2v重组蛋白的间接ELISA方法灵敏性可达1∶1280,批内变异系数小于4.15%,批间变异系数小于9.48%;建立的基于ASFV全病毒蛋白的间接ELISA方法灵敏性可达1∶2560,批内变异系数小于3.83%;批间变异系数小于5.04%。这两种方法均不与PRRSV、CSFV、PCV2等常见猪病阳性血清发生反应,特异性强。在96份临床猪血清样本中检出79份阴性血清、12份野毒株感染阳性血清和5份CD2v缺失株感染的阳性血清,与商品化试剂盒符合率分别达到87.5%与97.9%。本试验基于CD2v重组蛋白和ASFV全病毒蛋白建立了两种特异性强、敏感度高的间接ELISA方法,两者结合使用可以从血清学方法上区分ASFV野毒株感染和CD2v缺失株感染的临床样本,对非洲猪瘟的流行病学调查与精准防控提供了技术支持。The purpose of this study was to establish two indirect ELISA methods for the differential diagnosis of wild ASFV strains and ASFV(non-HAD) strains.The optimized EP402R gene sequence was used for prokaryotic expression of the recombinant protein.After purification,the protein was used as the antigen to establish an i ELISA method.The inactivated ASFV was purified by WOAH method and used as antigen to establish an i ELISA method.The sensitivity of the i ELISA method based on CD2v was 1 ∶ 1 280,with intra-batch CV less than 4.15%and inter-batch CV less than 9.48%.The sensitivity of the i ELISA method based on ASFV was 1 ∶ 2 560.The the intra-batch CV was less than 3.83%and inter-batch CV was less than 5.04%.The two methods did not react with PRRSV,CSFV,PCV2 and other porcine disease positive sera,and showed better specificity.Among 96 clinical pig serum samples,79 negative sera,12 positive sera and 5 positive sera infected with ASFV(non-HAD),the coincidence rate with commercial kits reached 87.5%and 97.9%.In this experiment,two indirect ELISA methods with better specificity and high sensitivity were established based on CD2v recombinant protein and ASFV.The combination of the two methods can distinguish clinical samples of wild ASFV and ASFV(non-HAD) infection from serological methods,providing technical support for the epidemiological investigation and precise prevention and control of African swine fever.

关 键 词：非洲猪瘟病毒 ELISA 鉴别诊断 

分 类 号：S852.659.1[农业科学—基础兽医学]