基于猪丁型冠状病毒重组蛋白N间接ELISA检测方法的建立与初步应用  

Establishment and preliminary application of indirect ELISA based on recombinant protein N for the detection of antibodies against porcine deltacoronavirus

作　　者：刘磊[1,2] 秦毅斌 陈忠伟[1] 赵武[1] 段群棚[1] 全琛宇[1] 赵硕 许心婷 陈婷婷 梁家幸 周英宁[1] 许艺兰[1] 李斌 蒋冬福[1] 卢敬专[1] 何颖 卢冰霞[1] LIU Lei;QIN Yi-bin;CHEN Zhong-wei;ZHAO Wu;DUAN Qun-peng;QUAN Chen-yu;ZHAO Shuo;XU Xin-ting;CHEN Ting-ting;LIANG Jia-xing;ZHOU Ying-ning;XU Yi-lan;LI Bin;JIANG Dong-fu;LU Jing-zhuan;HE Ying;LU Bing-xia(Guangxi Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning 530001,China;Guangxi Agricultural Vocational and Technical University,Nanning 530007,China)

机构地区：[1]广西兽医研究所广西兽医生物技术重点实验室,广西南宁530001 [2]广西农业职业技术大学,广西南宁530007

出　　处：《中国兽医科学》2022年第11期1347-1354,共8页Chinese Veterinary Science

基　　金：广西创新驱动发展专项资金项目(桂科AA17204057);玉林市科学研究与技术开发项目(玉市科20220515;玉市科20220516);柳州市科学研究与技术开发项目(2020NACB0804)。

摘　　要：为建立快速、准确的猪丁型冠状病毒(PDCoV)抗体ELISA检测方法,本研究以重组蛋白N作为包被抗原,建立了PDCoV间接ELISA抗体检测方法。结果显示,经条件筛选,确定了该ELISA最佳反应条件:重组蛋白每孔包被0.2μg;待检血清1∶80稀释,孵育60 min;酶标二抗1∶4000稀释,反应30 min;底物显色10 min。待检血清样品S/P值>0.200时判定为阳性,S/P值<0.173时判定为阴性,S/P值介于0.173和0.200之间则判为可疑。特异性试验结果表明,该检测方法不与HCV、FMDV、PRRSV、PRV、TGEV和PEDV的阳性血清发生交叉反应;重复性试验结果显示,批内变异系数为0.79%~4.75%,批间变异系数在1.92%~6.60%之间,表明重复性良好;敏感性试验结果显示,该方法可检测到血清最大稀释度为1∶320。符合率试验结果显示,本方法与中和试验检测结果符合度高,阳性符合率、阴性符合率和总符合率分别为86.36%、96.43%和92.00%。利用本研究建立的ELISA方法对456份采自广西地区不同阶段猪群的血清样品进行检测,结果显示,总体样品阳性率为40.40%,不同阶段猪群抗体阳性率有较大区别,后备母猪的阳性率可高达100%,保育猪阳性率最低仅为3.90%。Aim to establish a rapid and accurate ELISA method for the detection of PDCo V antibody,an indirect ELISA method for the detection of PDCo V antibody was optimized by using the purified recombinant protein N as the coating antigen.The results showed that the optimal conditions of ELISA as follows,the recombinant protein was coated with 0.2μg per well,the serum was diluted at 1∶80 and incubated for60 min,enzyme labeled secondary antibody was diluted at 1∶4000 and reacted for 30 min,finally add substrate for color development for 10 minutes under room temperature.The serum sample was determined to be positive while S/P value>0.200,to be negative when S/P value<0.173,to be suspected when S/P value between 0.173 and 0.200.Specificity experiment showed that the method did not cross-react with positive serums of HCV,FMDV,PRRSV,PRV,TGEV,and PEDV.Repeatability experiment showed good repeatability with inter-assay coefficients of variation ranging from 0.79%—4.75%and intra-assay coefficients of variation ranging from 1.92%—6.60%.Sensitivity experiment showed that the maximum dilution ratio of serum detected was 1∶320.Coincidence rate test showed that the method has high consistency with the results of neutralization test.The positive coincidence rate,negative coincidence rate and total coincidence rate are 86.36%,96.43%and 92.00%respectively.The indirect ELISA established in this study was used to detect 456 serum samples of pigs at different stages in Guangxi.The overall positive rate of samples was 40.40%,and the positive rates of pigs at different stages were quite different.The positive rate of gilts can be as high as 100%,and the lowest positive rate of nursery pigs was only 3.90%.

关 键 词：猪丁型冠状病毒 N蛋白 间接ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]