细胞培养和组织样本中虹彩病毒的定量。

Quantitation of ranaviruses in cell culture and tissue samples.

机构信息

Finnish Food Safety Authority Evira, Veterinary Virology Research Unit, Helsinki, Finland.

出版信息

J Virol Methods. 2011 Jan;171(1):225-33. doi: 10.1016/j.jviromet.2010.11.004. Epub 2010 Nov 16.

DOI:10.1016/j.jviromet.2010.11.004

Abstract

A quantitative real-time PCR (qPCR) based on a standard curve was developed for detection and quantitation of ranaviruses. The target gene for the qPCR was viral DNA polymerase (DNApol). All ten ranavirus isolates studied (Epizootic haematopoietic necrosis virus, EHNV; European catfish virus, ECV; European sheatfish virus, ESV; Frog virus 3, FV3; Bohle iridovirus, BIV; Doctor fish virus, DFV; Guppy virus 6, GV6; Pike-perch iridovirus, PPIV; Rana esculenta virus Italy 282/I02, REV282/I02 and Short-finned eel ranavirus, SERV) were detected with the qPCR assay. In addition, two fish cell lines - epithelioma papulosum cyprini (EPC) and bluegill fry (BF-2) - were infected with four of the isolates (EHNV, ECV, FV3 and DFV), and the viral quantity was determined from seven time points during the first three days after infection. The qPCR was also used to determine the viral load in tissue samples from pike (Esox lucius) fry challenged experimentally with EHNV.

摘要

建立了一种基于标准曲线的定量实时 PCR(qPCR)方法，用于检测和定量虹彩病毒。qPCR 的靶基因是病毒 DNA 聚合酶 (DNApol)。研究的十种虹彩病毒分离株（流行性造血器官坏死病毒、EHNV；欧洲鲶鱼病毒、ECV；欧洲七鳃鳗病毒、ESV；蛙病毒 3、FV3；博莱病毒、BIV；医生鱼病毒、DFV；孔雀鱼病毒 6、GV6；梭鲈虹彩病毒、PPIV；Rana esculenta 病毒意大利 282/I02、REV282/I02 和短鳍鳗虹彩病毒、SERV）均通过 qPCR 检测方法进行了检测。此外，用四种分离株（EHNV、ECV、FV3 和 DFV）感染了两种鱼类细胞系 - 鲤鱼上皮瘤细胞 (EPC) 和蓝鳃太阳鱼幼鱼 (BF-2)，并在感染后前三天的七个时间点测定了病毒数量。qPCR 还用于确定经实验性感染 EHNV 的欧白鲑幼鱼组织样本中的病毒载量。