大口黑鲈溃疡综合征病毒TaqMan-MGB探针荧光定量PCR检测方法的建立  

Development of a TaqMan-MGB Real Time PCR Assay for Largemouth Bass Ulcerative Syndrome Virus Detection

作　　者：马冬梅[1] 白俊杰[1] 邓国成[1] 李胜杰[1] 张莉莉[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所中国水产科学研究院热带亚热带鱼类选育与养殖重点开放实验室,广东广州510380

出　　处：《华南农业大学学报》2011年第2期99-102,共4页Journal of South China Agricultural University

基　　金：国家科技支撑计划(2006BAD01A1209);广东省科技计划项目(2007B020708008);农业部公益性行业科研专项(200903045)

摘　　要：根据大口黑鲈溃疡综合征病毒（Largemouth bass ulcerative syndrome virus,LBUSV）的甲基转移酶（MTase）基因核苷酸序列设计引物及TaqMan-MGB探针,优化荧光定量PCR反应条件和反应体系,建立了LBUSV的TaqMan-MGB探针荧光定量PCR检测方法.用含有MTase基因的质粒pDNA-MT系列稀释作为标准品模板,在荧光定量PCR仪上进行PCR反应,绘制标准曲线,斜率为-3.24,R2=0.999,呈现很好的线性关系,扩增效率1.04.灵敏度检测结果表明,PCR反应24个循环时可以检测到的质粒最小浓度是102拷贝数/μL.选用107、106、105、104拷贝数/μL 4个浓度梯度质粒做模板,变异系数在1.1%-3.4%范围内,说明该方法具有良好的重复性和稳定性.利用该方法可以准确地检测出感染LBUSV的病鱼,特异性强.Based on the nucleic sequence of DNA methyltransferase（MTase） gene from largemouth bass ulcerative syndrome virus（LBUSV）,primers and TaqMan-MGB probe were designed and synthesized to use for the detection of virus by real time PCR.After optimizing PCR annealing temperature and probe concentration,the fluorescence quantitative real time PCR method for LBUSV detection was developed.Series dilution of linear plasmid pDNA-MT as standard templates,PCR was performed on ABI 7300 PCR System.The standard curve was drawn,and the slope was-3.24（R2=0.999）,with a good linearity.The amplification efficiency of PCR reaction was calculated as 1.04.Sensitive assay showed that the method could detect 102 copies/μL plasmid templates within 24 PCR cycles.It is good repeatability and stability that the variation coefficient of the PCR ranged 1.1%-3.4%,for 107,106,105,104 copies/μL plasmid as PCR templates.The method showed good specialty and detected naturally LBUSV-infected largemouth bass,with precision.

关 键 词：大口黑鲈溃疡综合征病毒 TAQMAN-MGB探针 定量PCR 病毒检测 

分 类 号：S94[农业科学—水产养殖]