基于CA-TM融合蛋白的梅迪-维斯纳病毒胶体金免疫层析检测方法的建立与应用  

Establishment and application of colloidal gold immunochromatographic assay for detecting Maedi-Visna virus based on CA-TM fusion protein

作　　者：陈思旭 包磊 李慧萍[1,2,3] 张良 刘淑英 CHE Sixi;BAO Li;LI Huiping;ZHANG Liang;LIU Shuying(College of Veterinary Medicine,nner Mongolia A gricultural University,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease,Ministry of A griculture and Rural Affairs,Hohhot 010018,China;Inner Mongolia Key Laboratory of Basic Veterinary Science,Hohhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业农村部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018 [3]内蒙古自治区基础兽医学重点实验室,内蒙古呼和浩特010018

出　　处：《中国兽医科学》2024年第8期1002-1009,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(32072819,32360863);内蒙古科技重大专项计划项目(2021ZD0010);内蒙古草原英才创新团队项目(20151031);内蒙古教育厅兽医基础与牛羊疫病防控技术研发创新团队项目(NMGIRT2412);内蒙古自治区研究生科研创新项目(B20210174Z)。

摘　　要：旨在建立一种快速、准确的梅迪-维斯纳病毒(MVV)抗体的胶体金免疫层析检测方法。利用本实验室的重组CA-TM融合蛋白(r CA-TM)标记检测线,以鸡Ig Y抗体标记质控线,以重组链球菌蛋白(r-SPG)与鸡Ig Y用于胶体金标记。经各条件优化建立MVV胶体金免疫层析快速检测方法,并通过特异性、敏感性、稳定性以及临床应用对其检测效果进行评价。结果显示,成功表达、纯化并获得了r CA-TM;成功建立的胶体金免疫层析法特异性试验结果显示不能检测除MVV以外的如口蹄疫病毒、小反刍兽疫病毒、绵羊肺炎支原体、布鲁菌等绵羊常见病原;敏感性试验结果显示最低可检出2400倍稀释的MVV阳性血清;稳定性试验结果显示在模拟6个月避光常温密封保存后的试纸条仍具有良好的检测性能。利用建立的MVV胶体金检测法和广泛应用于MVV检测的进口商品化MVV抗体ELISA检测试剂盒对40份疑似病例血清样品进行对比检测,结果显示:用胶体金检测方法检出MVV抗体阳性血清17份,阴性血清23份;用进口商品化MVV抗体ELISA检测试剂盒检出阳性血清16份,阴性血清24份,二者相比整体符合率为97.5%。综上,成功建立了MVV胶体金免疫层析快速检测方法,该方法研制的检测试纸条性能良好、便于储存、适合现场快速检测和批量检测,对MVV的监测、防控有重要意义,为内蒙古地区MVV的净化提供了新的检测方案。The purpose of this study was to establish a rapid and accurate colloidal gold immunochromatographic method for the detection of Maedi-Visna virus(MVV)antibodies.The recombinant CA-TM fusion protein(r CA-TM)labeled detection line which has been authorized by our laboratory.The quality control line was labeled with chicken Ig Y antibody,and r-SPG and chicken Ig Y were used for colloidal gold labeling.The colloidal gold immunochromatography method for rapid detection of MVV was established after optimization of various conditions,and the detection effect was evaluated by specificity,sensitivity,stability and clinical application.In result,we successfully expressed,purified and obtained the colloidal gold immunochromatographic assay established by r CA-TM;the specificity test results of colloidal gold immunochromatography showed that it could not detect other common sheep pathogens such as foot-and-mouth disease virus,Peste des petits ruminant virus,Mycoplasma ovis and Brucella except MVV,and results of sensitivity test showed that MVV-positive serum with a minimum dilution of 2400times could be detected.The stability test results showed that the test strip still had good detection performance after 6 months of sealed storage at room temperature,and the MVV colloidal gold detection method and the imported commercial MVV antibody ELISA detection kit widely used in MVV detection were used to detect 40 serum samples of suspected cases.The results showed that 17 MVV antibody positive sera and 23 negative sera were detected by colloidal gold detection.The imported commercial MVV antibody ELISA detection kit detected 16 positive sera and 24 negative sera,with an overall coincidence rate of 97.5%.In summary,this experiment successfully established a rapid detection method of MVV colloidal gold immunochromatography.The test strip developed by this method has good performance,is easy to store,and is suitable for on-site rapid detection and batch detection.It is of great significance for the monitoring,prevention and control

关 键 词：梅迪-维斯纳病毒 原核表达 融合蛋白 胶体金 免疫层析 

分 类 号：S852.659.3[农业科学—基础兽医学]