犬细小病毒时间分辨免疫荧光分析方法的建立及初步应用  

Establishment and preliminary application of time-resolved immunofluorescence analysis method for Canine parvovirus

作　　者：陈翠翠 梁焕坤 赖宏锐 钟树海 黎杰星 李来庆 CHEN Cui-cui;LIANG Huan-kun;LAI Hong-rui;ZHONG Shu-hai;LI Jie-xing;LI Lai-qing(Guangzhou Youdi Biotechnology Co.,Ltd.,Guangzhou 510663,China)

机构地区：[1]广州优迪生物科技股份有限公司,广东广州510663

出　　处：《中国预防兽医学报》2021年第2期165-170,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：广州市科技计划项目珠江新星专题(201906010055)。

摘　　要：为建立犬细小病毒(CPV)的时间分辨免疫荧光分析(TRFIA)方法并制备试剂盒,本实验采用抗CPV单克隆抗体(MAb)7D5作为包被抗体,Eu3+标记的MAb 2F7作为检测抗体,对各反应条件优化后制备双抗体夹心TRFIA试剂盒。并评价了其灵敏度、准确度、特异性、重复性和稳定性。利用该试剂盒与RT-PCR方法同时检测了疑似患病犬的临床粪便、呕吐物、血液样品(各60份)和CPV阴性犬这3种临床样品各10份,比较并计算二者的符合率。优化结果显示,最佳的TRFIA反应条件为:MAb 7D5的包被浓度为15μg/mL;Eu^(3+)标记试剂与标记MAb 2F7(2.5 mg/mL)最佳体积分别为70μL和30μL;反应体系为25μL CPV标准品或待测样品+200μL分析缓冲液+200μL Eu^(3+)-检测抗体+200μL增强液。灵敏度试验显示TRFIA方法对CPV灭活病毒检测灵敏度为0.83 ng/mL。准确度试验结果显示,不同浓度CPV标准品稀释(1:2、1:4、1:8)后的稀释回收率在89.25%~100.8%;特异性试验结果显示,该方法除对不同亚型CPV的检测结果均为阳性外,对犬瘟热病毒、犬副流感病毒、犬腺病毒I型、犬冠状病毒、猫细小病毒、水貂肠炎病毒等检测均为阴性,无明显交叉反应;对高、中、低3种不同浓度CPV的重复性试验结果显示,批内变异系数为2.34%~4.76%,批间变异系数为2.38%~5.10%;稳定性试验结果显示,该试剂盒至少可在4℃稳定保存6个月,37℃保存7 d;临床样品的检测结果显示,利用该方法从大部分疑似患病犬的粪便、血液以及呕吐物中均检测到了CPV,而CPV阴性犬这3类样品的检测结果均为阴性。本研究建立的TRFIA法和RT-PCR法检测结果一致,符合率达到100%。上述结果表明,本研究制备的CPV TRFIA试剂盒灵敏度准确度高、特异性强、重复性、稳定性好,且可检测的临床样品种类多,为临床样品的检测提供有效技术手段。To establish a time-resolved immunofluorescence analysis(TRFIA)method for Canine parvovirus(CPV)and prepare the corresponding kits anti-CPV monoclonal antibody(MAb)7D5 as the coating antibody and Eu3+labeled MAb 2F7 as the detection antibody were used to prepare the double antibody sandwich TRFIA kit following the the optimization of reaction conditions,and the sensitivity,accuracy,specificity and repeatability of the kit,were evaluated in this study.3 different types of clinical stool,vomit,blood samples(60 samples each)of suspected cases and 10 samples from CPV-negative dogs were detected parallelly by the TRFIA kit and RT-PCR method,and their coincidence rate was compared based on the detection results.The final reaction conditions were optimized and as follows:coating antibody concentration(15μg/mL);the volumes of detection antibodies(2.5 mg/mL)and Eu^(3+)chelates were 70μL and 30μL,respectively;the reaction system(25μL CPV standard or test sample+200μL analysis buffer+200μL Eu^(3+)labeled antibody+200μL enhancement solution).Sensitivity test showed that the TRFIA method had a sensitivity of 0.83 ng/mL for the detection of CPV inactivated virus.Accuracy test results showed that the dilution recovery rate is 89.25%-100.8% at serial dilution(1:2,1:4 and 1:8);specificity test results showed that the results were all negative when CDV,CPIV,CAV-1,CCV,FPV,MEV were detected by the test kit without obvious cross-reaction;the results of repeatability test at high,medium and low dilution of CPVs showed that intra-batch coefficients of variation(CV)is between 2.34%and 4.76%,and inter-batch CV is between 2.38% and 5.10%;stability test results showed that the kit could be stably stored at 4℃ for 6 months and at 37℃ for 7 days;clinical sample test showed that VPV was positive from most of clinical stool,vomit,blood samples of suspected cases when detected by the TRFIA method,while the relevant samples from the CPV-negative dogs were negative.The detection results of established TRFIA method were consistent with

关 键 词：犬细小病毒 双抗体夹心 时间分辨免疫荧光分析 

分 类 号：S852.65[农业科学—基础兽医学]