基因Ⅱ型草鱼呼肠孤病毒TaqMan荧光定量PCR检测方法的建立与应用  

Establishment and application of a TaqMan real-time PCR assay for detection of grass carp reovirus genotype Ⅱ

作　　者：黄琦雯[1] 王庆[1] 王英英[1] 李莹莹[1] 石存斌[1] 任燕[1] 高彩霞 吴杰兴 郑树城[1] 曾伟伟[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/广东省水生动物免疫重点实验室,广东广州510380

出　　处：《中国预防兽医学报》2017年第10期804-809,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：广东省省级科技计划项目(2015A020209033);广东省省级科技计划项目(2016B020234003);广东省渔港建设和渔业发展专项(B201601-08);大宗淡水鱼产业技术体系(CARS-46-09)

摘　　要：基因Ⅱ型草鱼呼肠孤病毒(GCRV-Ⅱ)是当前引起草鱼出血病暴发与流行的主要病原,为建立快速检测GCRV-Ⅱ的方法,本研究根据GCRV-ⅡRd Rp基因的保守区域序列设计引物及TaqMan探针,经反应参数优化,建立了检测GCRV-Ⅱ的荧光定量PCR方法。结果显示,该方法仅对GCRV-Ⅱ的靶基因序列进行扩增,与其它非靶目标核酸均无交叉反应;其最低检出量为3拷贝/μL病毒核酸,比普通PCR的敏感度高100倍;组内和组间重复试验变异系数均小于1%。采用该方法检测60份草鱼出血病疑似样品,GCRV-Ⅱ阳性检出率为75%(45/60),与细胞分离结果一致。应用该方法分析了GCRV-Ⅱ在不同细胞和不同培养方式下病毒的增殖含量,结果显示GSB和PSF细胞增殖量最大,达106拷贝/μL^107拷贝/μL病毒核酸,转瓶接种比常规的细胞培养瓶和培养板接种其病毒含量低2个数量级。本研究建立的GCRV-ⅡTaqMan荧光定量PCR方法具有高效、特异、敏感、可重复性强的优点,适用于GCRV-Ⅱ的临床快速检测和病毒定量分析。The predominant genotype currently circulating in China is grass carp reovirus II （GCRV-II）, which is the main pathogen responsible for grass carp hemorrhage disease outbreaks. To establish the rapid and sensitive assay for GCRV-II detection, a TaqMan real-time PCR was developed with a pair of primers and probe designed according to the conserved sequence of the GCRV- II RdRp gene. The assay was specific to detect GCRV-II, but had no amplification from the DNA of other pathogens. The sensitivity of the method for GCRV-II was 3 copies/μL, which was 100 times sensitive than that of conventional PCR. The reproducibility tests in intra-assay and inter-assay indicated that the coefficients of variation were both less than 1%. 60 suspected samples of grass carp hemorrhagic disease were detected, of which 45 were positive for GCRV-II（positive rate 75%）, which were consistent with the results of cell separation. GSB and PSF cells were more suitable for proliferation of GCRV-II, and the virus titers could reach 106 copies/μL-107 copies/μL. The virus titers of inoculated with rollerbottle was about 100 times lower than conventional cell culture flasks and culture plates. To conclude, this method was efficient, specific, sensitive, accurate and suitable for the rapid detection and quantitative analysis of all the GCRV genotype II.

关 键 词：草鱼呼肠孤病毒 基因Ⅱ型 TAQ Man荧光定量PCR 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]