草鱼呼肠孤病毒RT-PCR检测方法的建立及临床应用  

Development and clinical application of RT-PCR method for grass carp reovirus

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

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

出　　处：《浙江农业学报》2012年第2期213-216,共4页Acta Agriculturae Zhejiangensis

基　　金：国家现代农业产业技术体系建设专项(CARS4631)

摘　　要：为了解草鱼呼肠孤病毒(Grass carp reovirus,GCRV)的感染流行情况及早期临床诊断的需要,研究并建立了该病毒的RT-PCR检测方法。根据GenBank中GCRV VP6蛋白基因保守序列设计引物,以GCRV弱毒疫苗株GCHV-892总RNA为模板,RT-PCR反应扩增出712 bp的目的条带,并对PCR反应体系和反应程序进行了优化,测定了该方法的敏感性和特异性。敏感性试验结果表明以含VP6蛋白基因的重组质粒为模板,该PCR方法的最低检测限为102copies.μL-1,特异性试验表明该引物仅能扩增GCRV核酸,而与白斑综合征病毒(WSSV)、嗜水气单胞菌(Aeromonas hydrophila)、病毒性神经坏死病毒(VNNV)无交叉反应性。应用所建立的RT-PCR方法检测了7份疑似出血病草鱼病样,3份病料获得了阳性扩增,随机挑选其中的1份阳性样品经克隆后测序,结果显示与GenBank中已知序列最高有99%的相似性。To understand the prevalence of Grass carp reovirus（GCRV）,and for the purpose of early clinical diagnosis,the viral RT-PCR detective method was established.Based on the VP6 gene sequences published in GenBank,a pair of specific primers was designed,and the total RNA of GCRV attenuated vaccine strain GCHV-892 was extracted as template to perform RT-PCR reaction,the obtained target band was 712 bp.Then the PCR reaction system and procedure were optimized,the sensitivity and specificity of the method were also considered.The results showed that the minimum detection limit of the sensitivity test was 102 copies·μL-1,in which the recombinant plasmid contained VP6 gene,was adopted as reaction template.The method has good specificity because no cross-reactivity was observed between white spot syndrome virus（WSSV）,Aeromonas hydrophila and viral nervous necrosis virus（VNNV）.Seven grass carp suspected with hemorrhagic disease were detected by the established assay,and three samples got positive amplification,then one of the positive specimens was cloned and sequenced,the results indicated that the sequence possesses maximum 99% similarity with known sequences in GenBank.

关 键 词：草鱼出血病 呼肠孤病毒 RT-PCR 检测方法 

分 类 号：S858.23[农业科学—临床兽医学]