水貂阿留申病毒的微滴式数字PCR方法的建立及初步应用  

Establishment and application of droplet digital PCR for detection of aleutia mink disease virus

作　　者：蔡晓庆 姜世金[2] 张金叶 孙圣福 王贵升 CAI Xiao-qing;JIANG Shi-jin;ZHANG Jin-ye;SUN Sheng-fu;WANG Gui-sheng(Animal Disease Prevention and Control Center of Shandong Province,Jinan 250100,China;Shandong Agricultural University,Taian 271018,China;Beijing Huadu Yukou Poultry Industry Co.,Ltd.,Beijing 101200,China;Weifang Animal Disease Prevention and Control Center,Weifang 261061,China)

机构地区：[1]山东省动物疫病与预防控制中心,山东济南250100 [2]山东农业大学,山东泰安271018 [3]北京市华都峪口禽业有限责任公司,北京平谷101200 [4]潍坊市动物疫病预防控制中心,山东潍坊261061

出　　处：《中国预防兽医学报》2024年第2期164-170,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：山东省现代农业产业技术体系特种经济动物产业疫病防控岗位(SDAIT-21-5)。

摘　　要：为建立水貂阿留申病毒(AMDV)微滴式数字PCR (ddPCR)检测方法,本研究根据AMDV VP2基因的保守区设计特异性引物和探针,通过PCR扩增AMDV VP2基因并克隆至p MD19-T载体,构建重组质粒p MD19-T-AMDV,并经PCR和测序鉴定后利用该质粒标准品作为模板,通过各反应条件的优化,初步建立了检测AMDV的ddPCR方法。分别以AMDV、水貂肠炎细小病毒、犬瘟热病毒、犬细小病毒、水貂流感病毒基因组为模板,按照本研究建立的ddPCR方法检测,评估其特异性;将1.43×10^(7)拷贝/μL~1.43×10^(0)拷贝/μL的重组质粒标准品作为模板,采用优化后的ddPCR和文献中的荧光定量PCR (qPCR)检测,评估该方法的敏感性;分别以不同浓度的重组质粒标准品作为模板,采用优化后的ddPCR分别进行批内和批间重复性试验,以变异系数(CV)评估该方法的重复性。优化结果显示,该方法的最佳引物终浓度为400 nmol/L (1.2μL)、探针终浓度为200 nmol/L (0.6μL)、退火温度为60℃。特异性试验结果显示,该方法仅可检测到AMDV和质粒标准品,而其他相关病毒均为阴性结果。敏感性试验结果显示,该方法对重组质粒标准品的检测限为1.43拷贝/μL,qPCR对质粒标准品的检测限为1.43×10^(2)拷贝/μL,前者比后者的敏感性高100倍。重复性试验结果,批内和批间重复性试验的变异系数均小于3%。对采集的70份病貂肝脏、脾、肾脏、环境拭子、肛拭子样品进行ddPCR和qPCR检测,结果显示,ddPCR对AMDV的检出率为24.28%(17/70);qPCR的检出率为15.7%(11/70),二者的阳性符合率为64.7%,阴性符合率为89.83%,总符合率为91.42%。上述结果表明,本研究建立的ddPCR方法特异性强、敏感性高和重复性好,可以检测各种临床样品及含量极低样品中的AMDV,为AMDV早期感染的检测、流行病学调查及AMD的防控提供有力技术手段。To establish a microdroplet digitalPCR(ddPCR) detection method for the aleutian mink disease virus(AMDV),this study designed specific primers and probes based on the conserved region of the AMDV VP2 gene.The AMDV VP2 gene was amplified byPCR and cloned into the pMD19-T vector.The recombinant plasmid pMD19-T-AMDV,identified byPCR and sequencing,was the plasmid standard used as a template.A preliminary ddPCR method for detecting AMDV was established by optimizing various reaction conditions.AMDV,mink enteritis parvovirus,canine distemper virus,canine parvovirus,and mink influenza virus were used to evaluate the specificity of the established ddPCR method.Using 1.43 ×10^(7) copies/μL-1.43 ×10^(0) copies/μL of the recombinant plasmid as a template,the sensitivity of this method was evaluated using optimized ddPCR detection and fluorescence quantitativePCR(qPCR) detection in literature.Inter-and intra-batch repeatability experiments were conducted.The coefficient of variation(CV value) was used to evaluate the effect of repeatability on this method.The optimization results showed that this method's optimal primer final concentration is 400nmol/L(1.2μL).The final concentration of the probe is 200nmol/L(0.6μL),and the annealing temperature is 60℃.The specificity test showed that this method can detect AMDV and the plasmid standard,while other related viruses showed negative results.The sensitivity test showed that the detection limit of this method for the plasmid standard is 1.43 copies/μL.The detection limit of the qPCR for plasmid standard is 1.43 ×10^(2) copies/μL.The former has 100 times higher sensitivity than the latter.The variation coefficient for intra-and inter-batch repeatability tests was less than 3%.Seventy samples of liver,spleen,kidney,environmental swab,and anal swab collected from diseased minks were subjected to ddPCR and qPCR detection.The results showed that the detection rate of AMDV by ddPCR was 28.33%(17/70).In contrast,the detection rate of qPCR is 15.7%(11/70).The positive agreement

关 键 词：微滴式数字PCR 检测 水貂阿留申病毒 

分 类 号：S852.65[农业科学—基础兽医学]