Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR
Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR
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DOI：
10.1016/j.jviromet.2005.10.005
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发表时间：
2006-03-01
影响因子：
3.1
通讯作者：
Perez, L
中科院分区：
文献类型：
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作者：
Chico, V;Gomez, N;Perez, L
建立了实时定量逆转录聚合酶链式反应(Q-RT-PCR)检测实验感染虹鳟鱼各脏器中出血性败血症病毒的方法。设计了针对病毒糖蛋白(G)和核蛋白(N)基因的引物和TaqMan探针。对细胞培养的病毒样品进行了Q-RT-PCR的效率、线性范围和检出限的评估。VHSV N基因扩增比VHSV G扩增更有效、更敏感。在细胞培养培养的病毒上，样本可以在每个反应的7个感染颗粒的对数范围内进行准确的分析。为了验证Q-RT-PCR在体内的有效性，进行了水浴感染试验，并采集了鱼的脾、肾、肝和血液样本，进行了VHSV的检测。Q-RT-PCR法是一种比常规RT-PCR法或细胞培养法更可靠的病毒诊断方法。本文介绍了在感染后不久的鱼中检测VHSV RNA的结果，以及在实验攻击后几周无症状的鱼的检测结果。这是首次报道Q-RT-PCR在体外和体内检测和定量VHSV的应用。本文还讨论了该方法在检测抗病毒治疗效果方面的适用性。(C)2005 Elsevier B.V.保留所有权利。
A quantitative real-time RT-PCR (Q-RT-PCR) was developed to detect and determine the amount of viral hemorrhagic septicemia virus (VHSV) in organs of experimentally infected rainbow trout. Primers and TaqMan probes targeting the glycoprotein (G) and the nucleoprotein (N) genes of the virus were designed. The efficiency, linear range and detection limit of the Q-RT-PCR were assessed on cell cultured virus samples. VHSV N gene amplification was more efficient and more sensitive than the VHSV G amplicon. On cell culture grown virus, samples could be accurately assayed over a range of seven logs of infectious particles per reaction. To demonstrate the utility of Q-RT-PCR in vivo, bath infection trials were carried out and samples from fish spleen, kidney, liver and blood were harvested and tested for VHSV. Q-RT-PCR was a more reliable method than either conventional RT-PCR or the cell culture assay for virus diagnosis. Results of VHSV RNA detection in fish shortly after infection as well as on asymptomatic fish several weeks after experimental challenge are presented here. This is the first report showing the utility of Q-RT-PCR for VHSV detection and quantitation both in vitro and in vivo. The suitability of this method to test the efficacy of antiviral treatments is also discussed. (c) 2005 Elsevier B.V. All rights reserved.