作　　者： ; ; ; ; ; ; ;

机构地区： 中国水产科学研究院珠江水产研究所

出　　处： 《中国水产科学》 2012年第2期329-335,共7页

摘　　要： 草鱼出血病是危害中国淡水养殖最为重要的病害之一,其病原为草鱼呼肠孤病毒(grass carp hemorrhagevirus,GCRV),其中,HZ08株是当前引起草鱼出血病的主要流行毒株。为建立一种快速、灵敏、特异的草鱼呼肠孤病毒主要流行株检测方法,本研究利用草鱼呼肠孤病毒HZ08株S7基因保守区,设计了一对能特异性扩增154 bp片段的引物和TaqMan探针,用含有S7基因全长的重组质粒PAVX1-S7作为标准品,构建质粒拷贝数与CT值的标准曲线,并对该方法的特异性、可重复性、敏感度进行评价。结果显示:标准曲线在6.0×1010～6.0拷贝数之间有很好的线性关系(r=0.999);实时荧光定量PCR最少可检测到6个阳性质粒,有较高的敏感性;试验内及试验间变异系数分别为0.82%与0.41%～0.52%,重复性强;对水生动物其他病毒均无扩增反应,具有很好的特异性。应用该方法对采集的32份草鱼出血病样品进行检测,其中28份为阳性,而以常规RT-PCR检测同样的样品,仅23份为阳性。本研究建立的草鱼呼肠孤病毒流行株实时荧光定量PCR检测方法在特异性、灵敏度、重复性方面具有较好的测试结果,在GCRV的快速检测和病毒初步定量中应用前景乐观。 Outbreaks of grass carp hemorrhage have caused widespread economic loss to the freshwater aquacul-ture industry in China.The disease is caused by grass carp reovirus（GCRV）.Currently,strain HZ08 is responsible for the majority of outbreaks of grass carp hemorrhage.We developed a rapid,efficient,and specific method for detection of GCRV epidemic causing strains.We designed a pair of specific primers and TaqMan probes targeting the HZ08 strain S7 gene.The standard curve was established using the standard template plasmid PVAX1-S7,which exhibited a corresponding relationship between the Ct and the copies of plasmid.We then evaluated the specificity,sensitivity,and repeatability of this approach.The linear relationship of copies was excellent within the range 6.0×1010-6.0（r=0.999 9）.Using FQ-PCR we were able to detect at least 6.0 copies of the S7 gene in the plasmid suggesting this method has high sensitivity.The coefficients of variation were 0.82 and 0.41%-0.52% for intragroup and intergroup,respectively,indicating that this method had high repeatability.Furthermore,the method had high specificity,based on the lack of amplification of other aquatic viruses.We tested 32 suspected grass carp hemorrhage specimens and returned 28 positive samples using FQ-PCR but just 23 positive samples using conventional RT-PCR.In summary,we developed a FQ-PCR method for the detection of HZ08 strain GCRV that has high specificity,sensitivity,and repeatability.Our method may also be applied to GCRV rapid detection and preliminary quantification.

关 键 词： 草鱼呼肠孤病毒 株 检测方法

领　　域： [农业科学] [农业科学]