Molecular double-check strategy for the identification and characterization of European Lyssaviruses
Molecular double-check strategy for the identification and characterization of European Lyssaviruses
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DOI：
10.1016/j.jviromet.2014.03.014
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发表时间：
2014-07-01
影响因子：
3.1
通讯作者：
Hoffmann, Bernd
中科院分区：
文献类型：
--
作者：
Fischer, Melina;Freuling, Conrad M.;Hoffmann, Bernd
狂犬病死后诊断的“金标准”是直接荧光抗体试验（FAT）。然而，在死前非神经样本材料或分解组织的情况下，FAT达到其极限，并且使用分子技术可能是有利的。在这项研究中，我们开发并验证了一种逆转录PCR级联协议可行的样本分类，即使是那些没有流行病学背景知识。这项研究的重点是最相关的欧洲狂犬病病毒。在第一步中，两个独立的N-和L-基因为基础的泛狂犬病病毒嵌入染料测定进行双重检查应用，以提高该方法的诊断安全性。对于第二步，通过两个独立的多重PCR系统对狂犬病病毒阳性样品进行表征。两种检测方法均为狂犬病病毒、欧洲蝙蝠狂犬病病毒1型和2型以及Bokeloh蝙蝠狂犬病病毒的基于探针的种属特异性多重PCR系统。所有检测方法均通过全面的狂犬病病毒阳性样本组以及来自不同宿主物种的阴性材料成功验证。这种双重检查策略允许对人类和动物的所有狂犬病病毒物种进行安全和灵敏的筛选、检测和表征，以及快速鉴定欧洲蝙蝠中目前未知的狂犬病病毒。(C)2014作者由Elsevier B. V.发布。这是CC BY许可下的开放获取文章
The "gold standard" for post-mortem rabies diagnosis is the direct fluorescent antibody test (FAT). However, in the case of ante-mortem non-neural sample material or decomposed tissues, the FAT reaches its limit, and the use of molecular techniques can be advantageous. In this study, we developed and validated a reverse transcription PCR cascade protocol feasible for the classification of samples, even those for which there is no epidemiological background knowledge. This study emphasises on the most relevant European lyssaviruses.In a first step, two independent N- and L-gene based pan-lyssavirus intercalating dye assays are performed in a double-check application to increase the method's diagnostic safety. For the second step, characterization of the lyssavirus positive samples via two independent multiplex PCR-systems was performed. Both assays were probe-based, species-specific multiplex PCR-systems for Rabies virus, European bat lyssavirus type 1 and 2 as well as Bokeloh bat lyssavirus. All assays were validated successfully with a comprehensive panel of lyssavirus positive samples, as well as negative material from various host species.This double-check strategy allows for both safe and sensitive screening, detection and characterization of all lyssavirus species of humans and animals, as well as the rapid identification of currently unknown lyssaviruses in bats in Europe. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license