Chi Yan-Yan, Tian Yuan-Yuan, Ye Xing, Deng Guo-Cheng, Li Jiong, Wang Hang-Jun
Pearl River Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou 510380, China.
Bing Du Xue Bao. 2011 Jul;27(4):358-65.
A strain of grass carp reovirus was isolated from sick grass carp with symptoms of haemorrhage in Guangdong province in 2009. The strain was tentatively named as GCRV-GD108 because it was isolated from grass carp and possessed 11 segments of dsRNA. Complete genome sequence analysis showed that significant differences existed between GCRV-GD108 and GCRV, as well as other known species of aquareovirus. In this study, molecular characteristics of diseased grass carp collected from different farms in Guangdong, Fujian and Hunan provinces were assayed. Based on the sequences of the 11 segments of GCRV-GD108, PCR primers corresponding to each of the segments were designed and synthesized. Total RNA of the diseased fish was extracted and used as templates of RT-PCR reaction. Specific amplification bands were obtained from all of the samples whereas no band was produced from GCRV standard strain. While using the primers specific to GCRV produced specific band in GCRV standard strain rather than in these collected samples. Sequencing of the amplification products showed that these samples displayed high similarities with each other (95.2%-99.4%), and they also shared high sequence similarities with that of GCRV-GD108 (95.0%-99.8%), suggesting that these samples shared similar molecular characteristics with those of GCRV-GD108, and were quite different from GCRV as well as the known species of genus aquareovirus. The results indicated that there are different molecular types of reovirus existed in the pond-cultured grass carp in China, and GCRV-GD108 is a representative strain in southern China, therefore great attention should be paid in order to control the disease efficiently, especially in vaccine preparation. Two pairs of primers were chosen to establish a duplex PCR assay method by combining each pair of the primers specific to GCRV-GD108 with the GCRV primer pair respectively. The duplex PCR assay method will enable the identification of GCRV-GD108 or GCRV by only a single PCR reaction.
2009年，从广东省出现出血症状的患病草鱼中分离出一株草鱼呼肠孤病毒。该毒株被暂命名为GCRV-GD108，因为它是从草鱼中分离出来的，并且拥有11个双链RNA片段。全基因组序列分析表明，GCRV-GD108与草鱼呼肠孤病毒以及其他已知的水生呼肠孤病毒种之间存在显著差异。在本研究中，对从广东、福建和湖南三省不同养殖场采集的患病草鱼的分子特征进行了检测。根据GCRV-GD108的11个片段的序列，设计并合成了对应于每个片段的PCR引物。提取患病鱼的总RNA并用作RT-PCR反应的模板。所有样品均获得特异性扩增条带，而GCRV标准毒株未产生条带。而使用针对草鱼呼肠孤病毒的引物在GCRV标准毒株中产生特异性条带，在这些采集的样品中则未产生。扩增产物的测序表明，这些样品彼此之间具有高度相似性（95.2%-99.4%），并且它们与GCRV-GD108也具有高度序列相似性（95.0%-99.8%），这表明这些样品与GCRV-GD108具有相似的分子特征，并且与草鱼呼肠孤病毒以及已知的水生呼肠孤病毒属物种有很大不同。结果表明，中国池塘养殖草鱼中存在不同分子类型的呼肠孤病毒，GCRV-GD108是中国南方的代表性毒株，因此为了有效控制该病，尤其是在疫苗制备方面，应予以高度重视。选择两对引物，分别将每对针对GCRV-GD108的引物与草鱼呼肠孤病毒引物对组合，建立了一种双重PCR检测方法。该双重PCR检测方法仅通过一次PCR反应就能鉴定出GCRV-GD108或草鱼呼肠孤病毒。
