大口黑鲈弹状病毒Taq Man荧光定量PCR检测方法的建立及初步应用  

Establishment and application of TaqMan qPCR assay for the detection of Micropterus salmoides rhabdovirus

作　　者：孔明慧 梁红茹[2] 李宁求[2] 林强[2] 牛银杰 罗霞[2] 马宝福 付小哲[2] KONG Ming-hui;LIANG Hong-ru;LI Ning-qiu;LIN Qiang;NIU Yin-jie;LUO Xia;MA Bao-fu;FU Xiao-zhe(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Key Laboratory of Fishery Drug Development,Ministry of Agriculture and Rural Affairs,Guangdong Provincial Key Laboratory of Aquatic Animal Immunology and Sustainable Aquaculture,Guangzhou 510380,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院珠江水产研究所农村农业部渔用药物创制重点实验室/广东省水产动物免疫技术与绿色养殖重点实验室,广东广州510380

出　　处：《中国预防兽医学报》2024年第4期379-384,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：广东省海洋经济发展(海洋六大产业)专项资金项目(粤自然资合[2022]41号);广东省乡村振兴战略专项资金项目(2022SPY00003);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金(2023TD48)。

摘　　要：为建立快速、敏感的大口黑鲈弹状病毒(MSRV)流行株Taq Man荧光定量PCR (qPCR)方法,本研究采用NCBI分析MSRV流行株N基因的保守序列,设计特异性引物和Taq Man探针,以构建的并经PCR和测序鉴定正确的重组质粒标准品p MD18-MSRV-N为模板,通过优化各反应条件初步建立MSRV的Taq Man q PCR检测方法。结果显示,建立的Taq Man q PCR方法标准曲线的R2为0.9906,质粒标准品在6.79×10^(8)拷贝/μL~6.79×10^(2)拷贝/μL范围内与Ct值均存在较好的线性关系。采用该方法检测MSRV及相关病毒,评估该方法的特异性,结果显示,该方法可以特异性检测MSRV,而草鱼出血病病毒、神经坏死病毒、鲤春病毒血症病毒、大口黑鲈双RNA病毒、鳜蛙虹彩病毒、传染性脾肾坏死病毒、石斑鱼虹彩病毒、大口黑鲈蛙虹彩病毒等8种常见鱼类病毒检测结果均为阴性;采用建立的方法检测不同浓度的质粒标准品(6.79×10^(8)拷贝/μL~6.79×10^(1)拷贝/μL),评估该方法的敏感性,结果显示,该方法的检测限为6.79×10^(2)拷贝/μL,比文献中的常规PCR方法敏感1 000倍;以不同批次或同一批次提取的不同浓度的质粒标准品作为模板,分别利用该方法检测,评估该方法的重复性,结果显示,该方法批内和批间重复性试验的变异系数均小于2%。采用该方法和文献中的常规PCR同时检测71份临床发病的大口黑鲈、鳜鱼、鲮鱼组织样品,结果显示阳性率为16.9%(12/71),阴性率为83.1%(59/71),且阳性样品均是从发病大口黑鲈的组织样品中检测到,而发病的鳜鱼、鲮鱼组织样品中均未检测到该病毒;常规PCR方法的阳性率为9.9%(7/71),阴性率为90.1%(64/71),二者的总符合率为93.0%。本研究建立的MSRV Taq Man q PCR方法特异性强、敏感性高、重复性和准确性均较好,可用于大口黑鲈临床样品的检测,为MSRV的快速检测提供了可行的技术手段。In order to establish a rapid and sensitive TaqMan qPCR method for the detection of Micropterus salmoides rhabdovirus(MSRV),in this study,NCBI was used to analyze the conserved sequences of N genes of MSRV epidemic strains.The correct constructed plasmid standard pMD18-MSRV-N was identified by PCR and sequencing.The TaqMan qPCR method for MSRV was preliminarily established by optimizing various conditions.Results showed that the R2 of the standard curve of the established TaqMan qPCR method was 0.9906,and the plasmid standard had a good linear relationship with the respective Ct values within the range of 6.79×10^(8) copies/μL to 6.79×10^(2) copies/μL.The specificity of the method was evaluated by detecting different viruses.This method can specifically detect MSRV.However,the test results of eight common fish viruses were negative,including grass carp reovirus(GCRV),nervous necrosis virus(NNV),spring viremia of carp virus(SVCV),largemouth bass birnavirus(LBBV),Siniperca chuatsi iridovirus,infectious spleen and kidney necrosis virus(ISKNV),singapore grouper iridovirus(SGIV)and largemouth bass ranavirus(LMBV).The sensitivity of the method was evaluated by detecting different concentrations of plasmid standards(6.79×10^(8) copies/μL to 6.79×10^(1) copies/μL).Results showed that the detectable limit of the method was 6.79×10^(2) copies/μL,which was 1000 times more sensitive than conventional PCR methods reported in the literature.The intra-assay and inter-assay repeatability of the method were evaluated by using different batches or different concentrations of plasmid standards extracted from the same batch as templates.The results showed that the coefficient of variation of the intra-assay and inter-assay repeatability of the method was less than 2%.The TaqMan qPCR and conventional PCR methods were used to detect seventy-one tissue samples from diseased Micropterus salmoides,Siniperca chuatsi,and dace.The results showed that the positive rate was 16.9%(12/71),and the negative detection rate was 83.1%(59/7

关 键 词：大口黑鲈弹状病毒 Taq Man荧光定量PCR N基因 

分 类 号：S941.6[农业科学—水产养殖]