基因Ⅰ型草鱼呼肠孤病毒TaqMan Real-Time PCR检测方法的建立及应用  

Establishment and application of a TaqMan Real-Time PCR assay for detection of grass carp reovirus genotype Ⅰ

作　　者：殷亮[1,2] 王庆[1] 曾伟伟[1] 李永刚[1,2] 王英英[1] 刘春[1] 梁红茹[1] 石存斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所 农业部渔药创制重点实验室 广东省水产动物免疫技术重点实验室,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2014年第4期570-576,共7页Journal of Fisheries of China

基　　金：国家科技支撑计划(2012BAD25B02);国家自然科学基金(31202026);水科院基本科研业务费(2014A06XK09)

摘　　要：为了建立和应用可检测基因I型草鱼呼肠孤病毒（GCRV）的荧光定量检测方法，实验根据基因I型GCRV的s6保守序列，分别设计扩增目的条带661bp普通引物（P1、P2）、扩增目的条带159bp荧光定量引物（F1、F2）和一条探针；同时构建含有目的片段的PVAX1-S6作为标准质粒，10倍梯度稀释构建质粒拷贝数与Ct值的标准曲线（Y=-3．389X＋38．076）。结果表明，此方法在3．9×10^7～3．9×10^1拷贝／uL之间相关性较好，R2为0．992，最小检测量为4拷贝／uL；且与基因Ⅱ型、Ⅲ型GCRV以及其他病原核酸无交叉反应。运用该方法检测16份草鱼出血病疑似样品，阳性结果为4份；而普通PCR检测仅2份显示阳性。本研究建立了针对基因I型GCRV的荧光定量检测方法，具有高效、特异、灵敏、可重复性强的优点，适合于目前基因I型GCRV的临床快速检测和病毒定量分析。In order to establish TaqMan Real-Time PCR for detection of GCRV genotype I ,the primers were designed according to conservative region of S6 as follows：normal primers designed as P1 and P2 with predicted product size of 661 bp,real time primers designed as F1 and F2 with predicted product size of 159 bp, and a probe. Standard curve （ Y = - 3. 389X ＋ 38. 076） was generated between the cycle threshold （ Ct ） and standard plasmid （PVAX1-S6） with 10-fold serial dilutions. The results showed that there was a good linear relationship between Ct value and template concentration with a detection range from 3.9 x 107 to 3.9 × 10^2 copies/uL, and the correlation coefficient reached 0. 992 while the detection limit of this method was 4 copies/uL for plasmid template. This assay had a specific detection of the GCRV genotype I , and had no detection signals to GCRV genotype II ,III and other pathogens. 16 suspected grass carp hemorrhage specimens were tested,and 2 more positive samples were detected using this established assay than normal RT-PCR. The developed TaqMan Real-Time PCR detection method for the GCRV genotype I with high efficiency, specificity, sensitivity and repeatability is available for clinical rapid detection and quantitative analysis of the GCRV genotype I.

关 键 词：草鱼呼肠孤病毒 基因I型 TAQMAN Real—Time PCR 定量分析 

分 类 号：S941.41[农业科学—水产养殖]