PANDAA for universal, pan-lineage molecular detection of filoviruses to enable rapid epidemic response.

埃博拉病毒[EBOV]和马尔堡病毒[MARV]是丝状病毒，可引起人类严重出血热和 并被列为世卫组织研发蓝图中优先考虑的紧迫病原体之一。基因组学 EBOV和MARV属的序列相差55%，并且在每个属内物种序列多样性 门槛是23%，因此基于黄金标准的qPCR方法的检测受到了显著的阻碍 丝状病毒之间的遗传变异。EBOV和MARV的临床诊断是困难的，因为早期阶段(即前 出血性)出现与一系列发热性疾病相关的非特异性症状，这些疾病表现为 受影响地区流行的其他传染病，如疟疾、黄热病、登革热、克里米亚-刚果 出血性(CCHFV)或拉沙热。为了隔离受感染的人，在EBOV或 只有以一致可靠的方式实施快速、准确的诊断，才能实现MARV疫情 性能。Aldatu率先使用了PANDAA技术，使基于探针的定量聚合酶链式反应能够用于靶标 通过同时适配和扩增不同的模板在高度可变的基因组区域中进行检测。PANDAA 独特地减少了遗传多态的存在，使原本不同的模板可以通过 荧光探头。因此，启用PANDAA的qPCR是普遍检测病原体的理想解决方案 显著的菌株、谱系和/或亚型序列多样性。阿尔达图具有独特的定位，可以提供快速的平移- 基于丝状病毒定量聚合酶链式反应的快速、灵敏的EBOV分子诊断方法 与现有的诊断方法相比，MARV具有更好的性能，不会受到基因变化的影响 新的病毒变种。PANDAA已经成功地应用于15个以上的亚型无关检测 HIV中的耐药突变(DRM)靶标。最近，我们开发了第一个拉沙热的泛系分析方法。 病毒(LASV)是世卫组织另一种具有高暴发潜力的优先病原体。我们建议利用独特的 PANDAA有能力开发一种快速、灵敏的丝状病毒检测分子诊断方法，并成为第一个 通过以下具体目标覆盖EBOV和MARV的泛物种：(1)发展泛物种-- 丝状病毒PANDAA分析，利用成熟的技术和专利PANDAA试剂设计；(2)分析性 验证包括确认物种包含性和高特异性；(3)PANDAA-丝状病毒的多重 用现有的LASV和CCHFV检测来产生病毒出血热(VHF)小组，包括 PANDAA-丝状病毒和PANDAA-VHF两种方法的热稳定性，以满足诊断要求 针对LMIC；(4)热稳定性PANDAA-丝状病毒和PANDAA-VHF分析和GMP的制造 (5)使用代表广泛的流通物种和地理位置的样本进行化验验证，具有多个 在疾控中心和世卫组织附属伙伴机构的参考实验室对试剂盒进行现场评估。作为第一个锅- 丝状病毒检测，我们将为所有地区提供快速、标准化的检测选项，这些地区可以使用Pre- 中心实验室现有的定量聚合酶链式反应设备，以从根本上改进诊断工作流程和防疫措施。

Ebolavirus [EBOV] and Marburgvirus [MARV] are filoviruses that cause severe hemorrhagic fevers in humans and primates and are listed among the urgently concerning pathogens prioritized in the WHO R&D Blueprint. Genomic sequences of the EBOV and MARV genera differ by >55%, and within each genera the species sequence diversity threshold is >23% thus detection by gold standard qPCR-based methods has been encumbered by significant genetic variability between filoviruses. Clinical diagnosis of EBOV and MARV is difficult as the early stages (i.e., pre- hemorrhagic) present with non-specific symptoms associated with a range of febrile illnesses that are displayed by other infectious agents endemic to affected areas e.g., malaria, yellow fever, dengue fever, Crimean-Congo hemorrhagic (CCHFV) or Lassa fevers. In order to isolate infected individuals, effective control during an EBOV or MARV outbreak can only be achieved by implementing rapid and accurate diagnostics with consistently reliable performance. Aldatu has pioneered the use of PANDAA technology, which enables probe-based qPCR for target detection in highly variable genomic regions by simultaneously adapting and amplifying diverse templates. PANDAA uniquely mitigates the presence of genetic polymorphisms to allow otherwise divergent templates to be detected by fluorescent probes. As such, PANDAA-enabled qPCR is an ideal solution for universal detection of pathogens with significant strain, lineage, and/or sub-type sequence diversity. Aldatu is uniquely positioned to deliver a rapid pan- filovirus qPCR-based assay that is rapid, sensitive molecular diagnostic for the detection and differentiation of EBOV and MARV with superior performance compared to existing diagnostics and won’t be affected by genetic changes in new viral variants. PANDAA has been successfully applied to subtype-independent detection of more than fifteen drug resistance mutation (DRM) targets in HIV. Recently, we developed the first pan-lineage assay for Lassa fever virus (LASV), another WHO priority pathogen with high outbreak potential. We propose to leverage the unique capabilities of PANDAA to develop a rapid, sensitive molecular diagnostic assay for filovirus detection, and the first with pan-species coverage of EBOV and MARV, through the following specific aims: (1) development of a pan- filovirus PANDAA assay, leveraging proven techniques and proprietary PANDAA reagent design; (2) analytical validation including confirmation of species inclusivity and high specificity; (3) multiplexing of the PANDAA-Filovirus assay with existing assays for LASV and CCHFV to produce a viral hemorrhagic fever (VHF) panel, including the thermostabilization of the both PANDAA-Filovirus and PANDAA-VHF assays, to meet the requirements of diagnostics targeted to LMICs; (4) GMP manufacturing of the thermostabilized PANDAA-Filovirus and PANDAA-VHF assays and (5) assay validation using samples representing a broad variety of circulating species and geographies, with multi- site evaluations of the test kits at reference labs at CDC- and WHO-affiliated partner institutions. As the first pan- filovirus assay, we will provide a rapid, standardized testing option for all regions that can be deployed using pre- existing qPCR equipment in central labs to radically improve the diagnostic workflow and epidemic preparedness.