Liu Zongxiao, Teng Yong, Liu Hong, Jiang Yulin, Xie Xiayang, Li Huifang, Lv Jiangqiang, Gao Longying, He Junqiang, Shi Xiujie, Tian Feiyan, Yang Jingshun, Xie Congxin
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, PR China; Show-how Bio-tech Institute, Beihai 536000, PR China.
J Virol Methods. 2008 Apr;149(1):103-9. doi: 10.1016/j.jviromet.2007.12.017. Epub 2008 Mar 4.
Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.
鲤春病毒血症病毒（SVCV）、传染性造血器官坏死病毒（IHNV）和病毒性出血性败血症病毒（VHSV）是三种重要的鱼类弹状病毒，可在野生和养殖鱼类中引发严重的世界动物卫生组织（OIE）分类疾病。在此，开发了一种新的多重实时定量RT-PCR（mqRT-PCR）检测方法，用于同时检测、鉴定和定量这三种弹状病毒。引物和探针组靶向SVCV糖蛋白（G）基因、IHNV核蛋白（N）基因和VHSV G基因的保守区域，并用于扩增。分析了mqRT-PCR检测方法的灵敏度、特异性和干扰试验。结果表明，该方法能检测到100拷贝的SVCV、220拷贝的IHNV和140拷贝的VHSV，使用梭子鱼幼鱼弹状病毒（PFRV）、传染性胰腺坏死病毒（IPNV）和草鱼呼肠孤病毒（GCRV）的RNA时无非特异性扩增和交叉反应。使用mqRT-PCR检测方法对80份临床鱼类样本进行了检测，并通过抗原捕获ELISA和细胞培养试验对结果进行了验证。该检测方法有潜力用于研究应用和诊断。
