草鱼呼肠孤病毒逆转录环介导等温扩增检测方法的建立  

Development of a Reverse Transcription Loop-mediated Isothermal Amplification(RT-LAMP) Method for Grass Carp Reovirus

作　　者：王晓丰[1] 薛晖[1,2] 丁正峰[1] 唐建清[1,2] 夏爱军[1,2] 熊怀生[3] 

机构地区：[1]江苏省淡水水产研究所,江苏南京210017 [2]国家大宗淡水鱼类产业技术体系南京综合试验站,江苏南京210017 [3]淮安市楚州区水产技术推广站,江苏淮安223001

出　　处：《福建农业学报》2012年第5期465-469,共5页Fujian Journal of Agricultural Sciences

基　　金：国家现代农业产业技术体系建设专项(CARS-46-31)

摘　　要：为更加快速、灵敏地检测草鱼呼肠孤病毒(Grass Carp Reovirus,GCRV),建立环介导等温扩增(RT-LAMP)检测方法。根据GenBank中GCRV HZ08等毒株VP6蛋白基因序列设计引物,以GCRV弱毒疫苗株(GCHV-892)总RNA为模板建立RT-LAMP反应,对扩增产物用限制性内切酶PvuⅡ进行酶切鉴定,所得酶切产物大小符合预期值。随后对LAMP反应体系包括Mg2+、dNTPs、甜菜碱(betaine)和内外引物浓度比进行了优化,并以含VP6蛋白基因的重组质粒(pEASY-VP6)为模板考量了该方法的敏感性,结果显示最低检测限为10copies.μL-1,比RT-PCR检测方法敏感10倍。特异性试验表明与白斑综合征病毒(WSSV)、嗜水气单胞菌(Aeromonas hydrophila)、病毒性神经坏死病毒(VNNV)无交叉反应。应用RT-LAMP方法检测16份疑似出血病草鱼,6份病料获得了阳性扩增,与RT-PCR检测结果一致。A RT-LAMP virus detection method was established for rapid, sensitive detection and early clinical diagnosis of grass carp reovirus （GCRV）. Based on the VP6 gene sequences of GCRV strain HZ08 from GenBank, a set of four specific primers was designed, and the total RNA of GCRV attenuated vaccine strain was extracted as template for RT-LAMP reaction. The amplification products were confirmed by Pvu Ⅱ restriction enzyme digestion. Then the LAMP reaction system, including Mg^2＋ , dNTPs, betaine, and the concentration ratio of inner to outer primers was optimized. The results of the sensitivity test using recombinant plasmid contained VP6 gene as template showed that the minimum detection limit is 10 copies ~ L-1 , a tenth the limit of RT-PCR. No cross-reactivity was observed among white spot syndrome virus （WSSV）, Aeromonas hydrophila and viral nervous necrosis virus （VNNV） in RT-LAMP. Sixteen suspected grass carp of hemorrhagic disease were detected by the established assay, and consistent with the RT-PCR result, six samples got positive amplification.

关 键 词：草鱼出血病 呼肠孤病毒 逆转录环介导等温扩增 检测方法 

分 类 号：S917[农业科学—水产科学]