Helsinki University and Helsinki University Hospital (HUSLAB), Department of Virology, Finland.
University of Helsinki, Department of Virology, Helsinki, Finland; Faculty of Veterinary Medicine, Department of Veterinary Biosciences, University of Helsinki, Finland.
J Clin Virol. 2019 May;114:26-31. doi: 10.1016/j.jcv.2019.03.010. Epub 2019 Mar 19.
During the five decades since their discovery, filoviruses of four species have caused human hemorrhagic fever outbreaks: Marburg (MARV) marburgvirus, and Zaire (EBOV), Sudan (SUDV) and Bundybugyo (BDBV) ebolaviruses. The largest, devastating EBOV epidemic in West Africa in 2014-16, has been followed by outbreaks of MARV in Uganda, 2017, and EBOV in Democratic Republic of Congo, 2018, emphasizing the need to develop preparedness to diagnose all filoviruses.
The aim of this study was to optimize a new filovirus RT-qPCR to detect all filoviruses, define its limits of detection (LOD) and perform a field evaluation with outbreak samples.
A pan-filovirus RT-qPCR targeting the L gene was developed and evaluated within the EbolaMoDRAD (Ebola virus: modern approaches for developing bedside rapid diagnostics) project. Specificity and sensitivity were determined and the effect of inactivation and PCR reagents (liquid and lyophilized format) were tested.
The LODs for the lyophilized pan-filovirus L-RT-qPCR assay were 9.4 copies per PCR reaction for EBOV, 9.9 for MARV, 1151 for SUDV, 65 for BDBV and 289 for Taï Forest virus. The test was set at the Pasteur Institute, Dakar, Senegal, and 83 Ebola patient samples, with viral load ranging from 5 to 5 million copies of EBOV per reaction, were screened. The results for the patient samples were in 100% concordance with the reference EBOV-specific assay.
Overall, the assay showed good sensitivity and specificity, covered all filoviruses known to be human pathogens, performed well both in lyophilized and liquid-phase formats and with EBOV outbreak clinical samples.
自发现以来的五十年中，四种病毒属的丝状病毒已导致人类出血热爆发：马尔堡（MARV）马尔堡病毒、扎伊尔（EBOV）、苏丹（SUDV）和本迪布焦（BDBV）埃博拉病毒。2014-16 年在西非发生的最大、最具破坏性的埃博拉疫情之后，2017 年乌干达暴发了马尔堡病毒，2018 年刚果民主共和国暴发了埃博拉病毒，这强调了有必要开发准备工作来诊断所有丝状病毒。
本研究旨在优化一种新的丝状病毒 RT-qPCR，以检测所有丝状病毒，确定其检测限（LOD），并使用暴发样本进行现场评估。
在 EbolaMoDRAD（埃博拉病毒：开发床边快速诊断的现代方法）项目中，开发并评估了针对 L 基因的泛丝状病毒 RT-qPCR。测定了特异性和敏感性，并测试了失活和 PCR 试剂（液体和冻干形式）的影响。
冻干泛丝状病毒 L-RT-qPCR 检测限为 9.4 个拷贝/PCR 反应，用于 EBOV；9.9 个拷贝/PCR 反应，用于 MARV；1151 个拷贝/PCR 反应，用于 SUDV；65 个拷贝/PCR 反应，用于 BDBV；289 个拷贝/PCR 反应，用于 Taï 森林病毒。该试验在塞内加尔达喀尔巴斯德研究所进行，共筛选了 83 份病毒载量范围为 5 至 500 万 EBOV 拷贝/反应的埃博拉患者样本。患者样本的结果与参考 EBOV 特异性检测完全一致。
总体而言，该检测具有良好的敏感性和特异性，涵盖了所有已知的人类病原体丝状病毒，在冻干和液体形式以及埃博拉暴发临床样本中均表现良好。
