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

PANDAA 用于丝状病毒的通用、全谱系分子检测，以实现快速流行病应对。

• 批准号: 10672434
• 负责人: Iain James MacLeod
• 金额: $ 93.12万
• 依托单位: ALDATU BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10672434
• 关键词: Affect; Area; Binding Sites; Biological Assay; Biological Sciences; Buffers; Certification; Collaborations; Consensus; Country; Crimean Hemorrhagic Fever; Custom; Dengue Fever; Detection; Development; Diagnostic; Diagnostic Reagent Kits; Disease Outbreaks; Drug resistance; Dryness; Ebola virus; Ensure; Equipment; Evaluation; Fever; Filovirus; Fluorescent Probes; Genetic; Genetic Polymorphism; Genome; Genomic Segment; Genomics; Geography; Goals; HIV; Hemorrhage; Human; Individual; Infectious Agent; Influenza; Institution; Lassa Fever; Lassa virus; Malaria; Marburgvirus; Methods; Molecular; Molecular Diagnostic Testing; Mutation; Pan Genus; Pathogen detection; Performance; Positioning Attribute; Primates; Process; Production; Quality Control; RNA; Rapid diagnostics; Reaction; Reagent; Reproducibility; Sampling; Serum; Site; Specificity; Standardization; System; Techniques; Technology; Testing; Validation; Variant; Viral; Viral Hemorrhagic Fevers; Work; Yellow Fever; accurate diagnostics; associated symptom; clinical diagnosis; design; detection assay; detection limit; epidemic preparedness; epidemic response; evaluation/testing; improved; in silico; innovation; low and middle-income countries; manufacture; molecular diagnostics; multiplex assay; novel; outbreak concern; pathogen; performance tests; priority pathogen; prototype; research and development; resistance mutation; thermostability

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.

埃博氏病毒[EBOV]和MARBURGVIRUS [MARV]是丝状病毒，引起人类和 灵长类动物并列在WHO R＆D蓝图中优先级的紧急关注的病原体中。基因组 EBOV和MARV属的序列不同于55％，在每个属中，物种序列多样性 阈值> 23％，因此通过基于黄金标准QPCR的方法检测到已有意义 丝状病毒之间的遗传变异性。由于早期阶段，EBOV和MARV的临床诊断很困难（即 出血）出现与一系列发热疾病有关的非特异性症状 其他感染性特工的内在对受影响区域的内在，例如疟疾，黄热病，登革热，克里米亚 - 康戈 出血（CCHFV）或LASSA发烧。为了隔离感染的个体，在EBOV期间有效控制或 只有通过实施始终可靠的快速，准确的诊断才能实现MARV爆发 表现。 Aldatu开创了Pandaa Technology的使用，该技术可实现基于探针的QPCR 通过简单地调整和放大不同模板，在高度可变的基因组区域中检测。熊达 独特地减轻遗传多态性的存在，以允许通过不同的模板检测到不同的模板 荧光问题。因此，支持熊猫的QPCR是与病原体普遍检测的理想解决方案 显着的应变，谱系和/或亚型序列多样性。 Aldatu的独特位置可以提供快速的泛滥 基于FILOVIRUS QPCR的测定，它是EBOV检测和分化的快速，灵敏的分子诊断 与现有诊断相比，MARV具有卓越的性能，不会受到遗传变化的影响 在新的病毒变体中。 Pandaa已成功地应用于超过15个以上的亚型检测 HIV中的耐药性突变（DRM）靶标。最近，我们开发了第一个用于Lassa Fever的Pan-Linege分析 病毒（LASV），另一位优先病原体具有高爆发潜力的病原体。我们建议利用独特的 熊达的能力开发用于filevirus检测的快速，敏感的分子诊断测定法，第一个 通过以下特定目的，通过泛型的EBOV和MARV覆盖范围：（1） filevirus pandaa分析，利用了经过验证的技术和专有熊猫试剂设计； （2）分析 验证，包括确认物种包容性和高特异性； （3）熊猫 - 菲洛尔病毒的多路复用 对LASV和CCHFV进行现有测定的测定，以产生病毒出血热（VHF）面板，包括 熊猫 - 菲洛尔病毒和熊猫-VHF分析的恒温化，以满足诊断的要求 针对LMIC； （4）GMP制造恒温pandaa-filovirus和Pandaa-VHF分析和 （5）使用代表各种循环物种和地理的样品进行测定验证，具有多种 在CDC和WHO相关合作伙伴机构的参考实验室的测试套件的现场评估。作为第一个泛 FILOVIRUS分析，我们将为所有可以使用Pre-pre-pre-extimions提供快速，标准化的测试选项 中央实验室中的现有QPCR设备从根本上改善了诊断工作流程和流行病的准备。