PANDAA for universal, pan-lineage molecular detection of Crimean-Congo Hemorrhagic Fever virus

克里米亚-刚果出血热(CCHF)是一种扁虱传播的疾病，在人类中死亡率很高，而且 有爆发的可能。CCHF分布在欧洲、亚洲和非洲的30个国家，具有极其广泛的 不断扩大的范围。其病原体CCHF病毒(CCHFV)被列为最受关注的病原体之一。 在世卫组织的研发蓝图中被列为优先事项。在至少有7个地区分支的情况下，用金标准qPCR- 以菌株为基础的方法受到与菌株相关的显著遗传变异性的阻碍。现有的CCHFV检测方法 通常仅限于区域使用，这对标准化和质量保证构成了障碍。世界卫生组织呼吁 作为全球优先事项，开发通用的CCHFV诊断方法。阿尔达图生物科学公司率先使用了这种技术 PANDAA技术的一部分，它使基于探针的qPCR能够通过以下方式在高度可变的基因组区域中进行靶标检测 同时适应和放大不同的模板。PANDAA独一无二地减少了目标的存在- 允许用通用荧光探针检测原本不同的模板的近端多态 也有类似的敏感性。因此，启用PANDAA的qPCR是普遍检测病原体的理想解决方案 具有显著的菌株、谱系和/或亚型序列多样性。阿尔达图生物科学公司的独特定位是 提供基于QPCR的快速泛谱系CCHFV诊断。PANDAA已成功应用于亚型- 独立检测HIV中15个以上的耐药突变(DRM)靶标。最近，我们完成了 首次建立了拉沙热病毒(LASV)的泛系分析方法，LASV是世卫组织另一种高度流行的优先病原体 有爆发的可能。我们建议利用PANDAA的独特能力来开发一种快速、敏感的 用于CCHFV检测的分子诊断试验，以及第一个具有泛谱系覆盖的方法，通过以下方式 具体目标：(1)开发泛系PANDAA-CCHFV检测方法，利用成熟的技术和 专利PANDAA试剂设计；(2)PANDAA-CCHFV分析验证，包括确认 (3)PANDAA-CCHFV检测的热稳定性，以满足 针对LMICs的诊断要求；(4)冻干PANDAA-CCHFV检测的临床验证，使用 代表广泛的循环分支和地理位置的临床样本；以及(5)对 在疾控中心和世卫组织附属伙伴机构的参考实验室提供的PANDAA qDx CCHFV检测试剂盒。作为第一个锅- 血统分析，PANDAA-CCHFV将为全球所有地区提供快速、标准化的检测选择 CCHFV的射程。这种新颖的泛谱系检测分析最终可以在任何流行地区部署 中心实验室中预先存在的定量聚合酶链式反应设备，和/或集成到带有样本的封闭式护理点系统中 从根本上改进CCHFV诊断工作流程的处理。

Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne disease with high mortality rates in humans and high outbreak potential. Found in 30 countries across Europe, Asia, and Africa, CCHF has an extremely widespread and growing range. Its causative agent, CCHF virus (CCHFV), is listed among the urgently concerning pathogens prioritized in the WHO R&D Blueprint. With at least seven regional clades, CCHFV detection by gold standard qPCR- based methods has been encumbered by significant strain-associated genetic variability. Existing tests for CCHFV are often limited to regional use, presenting a barrier to standardization and quality assurance. The WHO has called for the development of universal CCHFV diagnostics as a global priority. Aldatu Biosciences 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 target- proximal polymorphisms to allow otherwise divergent templates to be detected with consensus fluorescent probes with similar sensitivities. 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 Biosciences is uniquely positioned to deliver a rapid pan-lineage qPCR-based CCHFV diagnostic. PANDAA has been successfully applied to subtype- independent detection of more than fifteen drug resistance mutation (DRM) targets in HIV. Recently, we completed development of 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 CCHFV detection, and the first with pan-lineage coverage, through the following specific aims: (1) development of a pan-lineage PANDAA-CCHFV assay, leveraging proven techniques and proprietary PANDAA reagent design; (2) analytical validation of the PANDAA-CCHFV assay, including confirmation of clade inclusivity and high specificity; (3) thermostabilization of the PANDAA-CCHFV assay, in order to meet the requirements of diagnostics targeted to LMICs; (4) clinical validation of the lyophilized PANDAA-CCHFV assay, using clinical samples representing a broad variety of circulating clades and geographies; and (5) multi-site evaluation of the PANDAA qDx CCHFV test kit at reference labs at CDC- and WHO-affiliated partner institutions. As the first pan- lineage assay, PANDAA-CCHFV will provide a rapid, standardized testing option for all regions within the broad range of CCHFV. This novel, pan-lineage detection assay could ultimately be deployed in any endemic region on pre-existing qPCR equipment in central labs, and/or integrated into a closed, point-of-care system with sample processing to radically improve the CCHFV diagnostic workflow.