Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics
Development and preliminary evaluation of a multiplexed amplification and next generation sequencing method for viral hemorrhagic fever diagnostics
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DOI：
10.1371/journal.pntd.0006075
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发表时间：
2017-11-01
影响因子：
3.8
通讯作者：
Nitsche, Andreas
中科院分区：
文献类型：
--
作者：
Brinkmann, Annika;Erguenay, Koray;Nitsche, Andreas
我们描述了一种基于靶向扩增和下一代测序（NGS）鉴定病毒性出血热（VHF）药物的新方法的开发和评估，并评估了该方法在诊断中的可行性。方法用引物设计超高倍扩增板，扩增已知的vhf相关病毒变异及相关对照。在基于半导体的测序平台上，通过对黄热病病毒、裂谷热病毒、克里米亚-刚果出血热病毒、埃博拉病毒、朱宁病毒和基孔肯雅病毒的核酸进行序列量化，对该小组的性能进行评估。比较了直接NGS和靶向扩增-NGS。使用来自CCHF患者的临床标本，通过基于实时纳米孔测序的平台进一步测试该面板。多重引物面板包括两个引物池，分别有285对和256对引物，用于鉴定46种引起出血热的病毒，包括相关菌株的6130种遗传变异。计算机验证显示，该小组检测到目标病毒物种所有已知遗传变异的97%以上。检测到的病毒株具有很高的特异性和敏感性。靶向扩增确保了在病毒浓度最低（1-10个基因组当量）的标本中检测到病毒读段，并使所调查的所有病毒在背景上的特异性读段显著增加。在临床标本中，该面板能够在测序后10分钟内检测病原体及其特征，从样品到结果的时间不到3.5小时。结论通过靶向扩增进行病毒富集，然后进行NGS是一种适用于vhf诊断的策略，可适用于高通量或纳米孔测序平台，并可用于监测或疫情监测。
BackgroundWe describe the development and evaluation of a novel method for targeted amplification and Next Generation Sequencing (NGS)-based identification of viral hemorrhagic fever (VHF) agents and assess the feasibility of this approach in diagnostics.MethodologyAn ultrahigh-multiplex panel was designed with primers to amplify all known variants of VHF-associated viruses and relevant controls. The performance of the panel was evaluated via serially quantified nucleic acids from Yellow fever virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever (CCHF) virus, Ebola virus, Junin virus and Chikungunya virus in a semiconductor-based sequencing platform. A comparison of direct NGS and targeted amplification-NGS was performed. The panel was further tested via a real-time nanopore sequencing-based platform, using clinical specimens from CCHF patients.Principal findingsThe multiplex primer panel comprises two pools of 285 and 256 primer pairs for the identification of 46 virus species causing hemorrhagic fevers, encompassing 6,130 genetic variants of the strains involved. In silico validation revealed that the panel detected over 97% of all known genetic variants of the targeted virus species. High levels of specificity and sensitivity were observed for the tested virus strains. Targeted amplification ensured viral read detection in specimens with the lowest virus concentration (1-10 genome equivalents) and enabled significant increases in specific reads over background for all viruses investigated. In clinical specimens, the panel enabled detection of the causative agent and its characterization within 10 minutes of sequencing, with sample-to-result time of less than 3.5 hours.ConclusionsVirus enrichment via targeted amplification followed by NGS is an applicable strategy for the diagnosis of VHFs which can be adapted for high-throughput or nanopore sequencing platforms and employed for surveillance or outbreak monitoring.