检测鸡毒支原体抗体的间接ELISA方法和HI试验方法的建立及初步应用  

Establishment and Preliminary Application of Indirect ELISA Method and HI Test for Detection of Mycoplasma Gallisepticum Antibody

作　　者：陈杨 孟林春 郭梦娇 张成成 薄宗义 楚电峰 曹永忠 吴艳涛[1,2] 张小荣 CHEN Yang;MENG Linchun;GUO Mengjiao;ZHANG Chengcheng;BO Zongyi;CHU Dianfeng;CAO Yongzhong;WU Yantao;ZHANG Xiaorong(Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;International Research Laboratory of Agriculture and Agri-Product Safety,Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou 225009,China;State Key Laboratory of Genetically Engineered Veterinary Vaccines,Qingdao Yebio Biological Engineering Co.,Ltd,Qingdao 266114,China)

机构地区：[1]扬州大学兽医学院、江苏省动物重要疫病预防控制协同创新中心,扬州225009 [2]扬州大学农业科技发展研究院、教育部农业与农产品安全国际合作联合实验室,扬州225009 [3]青岛易邦生物工程有限公司动物基因工程疫苗国家重点实验室,青岛266114

出　　处：《畜牧兽医学报》2023年第5期2062-2072,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：财政部和农业农村部:国家现代农业产业技术体系资助项目(CARS-40-K16);国家重点研发计划“科技助力经济2020”重点专项(SQ2020YFF0426460);动物基因工程疫苗国家重点实验室开放课题(AGVSKL-ZD-202003);江苏高校优势学科建设工程资助项目(2018年)。

摘　　要：本研究旨在建立一种检测鸡毒支原体(Mycoplasma gallisepticum,MG)抗体的间接ELISA方法,结合HI试验,为MG感染的监测和净化提供一套有效的组合技术方案。以原核表达的VlhA 3.03重组蛋白(rVlhA)作为包被抗原,使用单一稀释法构建了关于血清抗体滴度与1∶500血清稀释度处S/P值的回归方程lg(抗体滴度)=1.257×lg(1∶500处S/P值)+3.709,R^(2)=0.9176,S/P临界值为0.32,临界滴度是1200。经验证,rVlhA-ELISA方法具有良好的特异性、重复性,最低能检出1∶2000稀释的阳性血清。选择国内MG分离株SH/2020-1作为血凝抑制抗原建立HI试验方法。使用rVlhA-ELISA方法联合HI试验进行血清样品检测,IDEXX-MG方法作为对照,借助RT-qPCR方法监测MG感染情况。结果表明rVlhA-ELISA方法和IDEXX-MG监测的血清抗体趋势与HI复核结果均一致;rVlhA-ELISA与HI联合判定结果与IDEXX-MG符合率可达91.53%。上述结果显示,本研究建立的rVlhA-ELISA方法和HI试验具有较好的临床应用价值,可以初步应用于MG感染的临床大规模、快速筛查并对结果进行复核。This study aimed at establishing an indirect-ELISA method for the titer detection of Mycoplasma gallisepticum(MG)antibodies in chicken,which could provide a set of effective technology for large-scale clinical monitoring of MG infection combined with HI test established.Prokaryotic-expressed protein VlhA3.03(rVlhA)was obtained as envelope antigen.The linear equation lg(antibody titer)=1.257×lg(S/P value at 1∶500)+3.709 of serum antibody titer and S/P value at 1∶500 was established by a single serum dilution test and the correlation coefficient(R^(2))was 0.9176.The cutoff value of negative and positive S/P was determined to be 0.32,and the corresponding titer is 1200.The rVlhA-ELISA showed good specificity and repeatability.The detection limit of MG positive sera is 1∶2000.An HI assay was also established using domestic MG isolate SH/2020-1 as hemagglutination inhibition test antigen.The combined rVlhA-ELISA method and HI test,and IDEXX-MG were compared in serum sample detection,with RT-qPCR method to monitor MG infection.The results showed that the trend of serum antibody monitored by rVlhA-ELISA and IDEXX-MG was consistent with the result of HI assay.The coincidence rate between combined rVlhA-ELISA and HI,and IDEXX-MG was 91.53%.These results indicated that the rVlhA-ELISA and HI test established in this study were beneficial to clinical application,which can be applied to large-scale and rapid clinical screening of MG infection and recheck the results.

关 键 词：鸡毒支原体 VlhA蛋白 抗体检测 间接ELISA 

分 类 号：S852.62[农业科学—基础兽医学]