犬细小病毒时间分辨荧光免疫法的建立及初步应用  

Establishment and preliminary application of time-resolved fluoroimmunoassay method for canine parvovirus

作　　者：李红[1] 刘成倩[1] 孙凤萍[1] 高骏[1] 陆娜云 梁洪宇 刘惠莉[1] 易建中[1] LI Hong;LIU Cheng-qian;SUN Feng-ping;GAO Jun;LU Na-yun;LIANG Hong-yu;LIU Hui-li;YI Jian-zhong(Institute of Animal Husbandry&Veterinary Science,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean,Shanghai 201306,China)

机构地区：[1]上海市农业科学院畜牧兽医研究所,上海201106 [2]上海海洋大学水产科学国家级实验教学示范中心,上海201306

出　　处：《中国兽医科学》2021年第8期976-982,共7页Chinese Veterinary Science

基　　金：城市犬猫主要人畜共患病监测和公共卫生安全风险评估[沪农科创字(2019)第3-3号];上海市农业科学院卓越团队计划[农科创2017(B-03)]。

摘　　要：随着人们物质生活水平不断提升,宠物犬的养殖数量不断增加,由犬细小病毒(CPV)引起的疾病的发病率也呈升高趋势。CPV是我国出入境口岸宠物检疫的重要病原之一,为提高检疫效率和准确率,本研究建立了犬细小病毒时间分辨荧光免疫分析(TRFIA)方法。本研究主要选用犬细小病毒特异性单克隆抗体(MAb)4C11和7D5分别作为包被抗体和检测抗体,检测抗体用镧系元素中的铕(Eu3+)标记,制备出双抗体夹心TRFIA分析法。结果显示,该方法灵敏度可达0.79 ng/m L;CPV标准品回收率为96.48%~105.17%,同步检测CCV、CDV、CAV-1、CPIV标准品,结果均为阴性;重复性检测发现批内和批间变异系数分别为4.78%~6.03%和4.23%~6.42%;4℃可保存6个月以上,37℃可保存7 d以上。临床检测结果与PCR方法一致。结果表明,该方法具有灵敏度高、稳定性好、特异性强、重复性好、操作便捷,受本底因素影响少等优点,适用于CPV的快速检测。且读卡方式多样,满足了不同实验条件的要求,具有较高的临床实用价值和推广价值。With the raising of citizen’s living standard,pet dog population is increasing continuously in the cities,which also brought about more incidences of dog diseases caused by canine parvovirus(CPV).CPV is one of the main pathogens to be detected in custom quarantine on pets.To improve the detection efficiency and accuracy,we developed the time-resolved fluoroimmunoassay(TRFIA) for canine parvovirus in the study.TRFIA double-antibody sandwich for detecting CPV was developed by employing anti-CPV monoclonal antibody clone(4 C11) as the coating antibody and another antibody clone 7 D5 that was labeled with the Eu3+as the detection antibody.The results showed that the detection sensitivity was0.79 ng/m L and the recovery rate of CPV standard was 96.48%—105.17%.The simultaneous detection of CCV,CDV,CAV-1 and CPIV standard samples were negative.Repeatability test showed that coefficients of variation(CV) of intra and inter assays were 4.78%—6.03% and 4.23%—6.42% respectively.The prepared reagents and coated plates can be stored at 4 ℃ for 6 months or at 37 ℃ for 7 days without obvious quality decrease.Detection of clinical sample with the method showed the same result as PCR method.Therefore the established TRFIA method has the advantages of high sensitivity,good stability,strong specificity and convenience,which facilitates CPV rapid detection.There are several ways to read the result,which meet the requirements of different test facilities,so the established method is highly valued for clinical use.

关 键 词：犬细小病毒 时间分辨荧光免疫层析 检测 

分 类 号：S852.659.2[农业科学—基础兽医学]