Ⅱ型草鱼呼肠孤病毒三种定量检测方法的比较与评价  

Comparison and evaluation of the three different methods in quantitative detection of GCRV-Ⅱ

作　　者：曾伟伟 王英英 尹纪元 汤亚方 李莹莹 王庆 高彩霞 任燕 刘春 石存斌 ZENG Wei-wei;WANG Ying-ying;YIN Ji-yuan;TANG Ya-fang;LI Ying-ying;WANG Qing;GAO Cai-xia;REN Yan;LIU Chun;SHI Cun-bin(Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Fishery Drug Development,Ministry of Agriculture/Guangdong Key Laboratory of Aquatic Animal Immune Technology,Guangzhou,510380,China;College of Fisheries,Tianjin Agriculural University,Tianjin 300384,China)

机构地区：[1]中国水产科学研究院珠江水产研究所,广东省水产动物免疫技术重点实验室,农业农村部渔药创制重点实验室,广州510380 [2]天津农学院水产学院,天津300384

出　　处：《淡水渔业》2019年第6期63-69,共7页Freshwater Fisheries

基　　金：广东省自然科学基金重点项目(2017B030311018);广东省现代农业产业技术体系创新团队建设专项资金(2019KJ119);大宗淡水鱼产业技术体系(CARS-45-17);Ⅱ型草鱼呼肠孤病毒灭活疫苗规模化生产关键技术研究(2019ZD0703);广东省促进经济高质量发展专项资金海洋经济发展项目(GDOE[2019]A36)

摘　　要：为了解决Ⅱ型草鱼呼肠孤病毒(grass carp reovirus genotypeⅡ,GCRV-Ⅱ)含量和滴度测定方法的选择问题,本研究利用荧光定量PCR(quantitative PCR,qPCR)、免疫过氧化物酶单层细胞试验(immunoperoxidase monolayer assay,IPMA)和间接免疫荧光试验(indirect fluorescent assay,IFA)三种方法来对GCRV-Ⅱ进行检测和含量测定,从特异性、敏感性、可靠性和检测结果的相关性来比较这三种方法对于GCRV-Ⅱ定量检测的差异。结果显示:qPCR、IPMA和IFA三种方法检测GCRV-Ⅱ的最低限分别为8.6、86和86拷贝/μL,qPCR的检测灵敏度比IPMA和IFA高一个数量级;特异性分析表明,qPCR、IPMA和IFA三种方法均只能检测出GCRV-Ⅱ型分离株,而其它病毒和未感染病毒的阴性对照细胞均为检测阴性,表明这三种方法均具有较高的特异性;3种方法分别检测60份已知临床样品,qPCR、IPMA和IFA的诊断敏感性和特异性分别为96.7%(29/30)和100%(30/30)、100%(30/30)和93.3%(28/30)及100%(30/30)和100%(30/30);三种方法对于GCRV-Ⅱ的定量检测结果中病毒拷贝数和病毒滴度之间具有较好的相关性,其拟合曲线分别为:Y(IPMA,TCID 50/mL)=23.629 X-536174(qPCR,拷贝/mL)(R 2=0.996)、Y(IFA,TCID 50/mL)=20.318 X-575062(qPCR,拷贝/mL)(R^2=0.995)和Y(IPMA,TCID 50/mL)=1.161 X-1176(IFA,TCID 50/mL)(R^2=0.999)。结果表明,GCRV-Ⅱ病毒滴度的测定,除了用IPMA和IFA外,也可以采用qPCR方法通过测定病毒的核酸拷贝数来测算其病毒滴度。Three different methods of fluorescence quantitative PCR(qPCR),immunoperoxidase monolayer assay(IPMA)and indirect fluorescent assay(IFA)were used to detect and quantitative analysis GCRV-Ⅱand the differences of these three methods were compared from the aspects of specificity,sensitivity,reliability and correlation of results in order to choose the suitable ethods for Grass Carp ReovirusⅡ(GCRV-Ⅱ)virus titer determination.The results showed that the minimum detection limits of qPCR,IPMA and IFA for GCRV-Ⅱ were 8.6 copies/μL,86 copies/μL and 86 copies/μL,respectively.The analytical sensitivity of qPCR was at least one log10 higher than that of IPMA and IFA.Specificity analysis showed that the qPCR,IPMA and IFA could only detect GCRV-Ⅱ isolates,while negative controls that infected with other viruses and uninfected cells were all negative,indicating that all the three methods had good analytical specificity for GCRV-Ⅱ.The three methods tested sixty clinical samples with known results.The diagnostic sensitivity of qPCR,IPMA and IFA were 96.7%(29/30),100%(30/30)and 100%(30/30),respectively.The diagnostic specificity of qPCR,IPMA and IFA were 100%(30/30),93.3%(28/30)and 100%(30/30),respectively.Quantitative results for GCRV-Ⅱ showed good correlation among the qPCR、IMPA and IFA,and the fitted curve were Y(IPMA,TCID 50/mL)=23.629 X-536174(qPCR,copies/mL)(R 2=0.996),Y(IFA,TCID 50/mL)=20.318 X-575062(qPCR,copies/mL)(R 2=0.995)and Y(IPMA,TCID 50/mL)=1.161 X-1176(IFA,TCID 50/mL)(R^2=0.999),respectively.Therefore,in addition to IPMA and IFA,the titer of GCRV-Ⅱvirus could also be determined by measuring the copy number of the nucleic acid of the virus by qPCR method.

关 键 词：草鱼呼肠孤病毒 荧光定量PCR 免疫过氧化物酶单层细胞试验 间接免疫荧光试验 病毒滴度 

分 类 号：S941.41[农业科学—水产养殖]