PHS2019-02 Omnibus Solic of the NIH, CDC, and FDA for SBIR Apps No Clinical Trial (Parent SBIR R43/4

直接从捕蚊器中现场检测虫媒病毒 火鸟生物分子科学有限责任公司。 摘要 蚊子是世界上最致命的生物。在美国，蚊子是西尼罗河病毒的传播媒介， 以及病毒到达拉丁美洲和非洲。在发展中国家，它们造成了数百万的 每年都有疾病和死亡。常规的杀虫剂喷洒并不是一个“解决办法”，因为它会导致杀虫剂抗药性， 遭到民众反对，并消灭益虫。因此，EPA，CDC，ECDPC及其 地球仪的同行们强烈建议在喷洒前进行监测。佛罗里达的法律要求。 在第一阶段，我们创造了（a）产生二氧化碳吸引蚊子的创新技术，（B） （c）数据显示收集的RNA在温度下是稳定的， 佛罗里达夏季条件下至少3天，（d）直接扩增捕获的病毒RNA的方法 而不需要处理蚊子或Qiagen RNA制备物，和（e）允许稳健的多重扩增的试剂。 这些可以改变虫媒病毒监测的科学和实践的规格吸引了TrakITNow， 仪器设计的创新者。它的获奖陷阱（Moskeet）计数和物种蚊子，做 除了从蚊子身上收集样本并确定这些蚊子是否携带虫媒病毒之外， 该第2阶段项目将重新配置和衡量第1阶段的创新，以便在第3阶段 在Moskeet上有两种操作架构（OA 1和OA 2）。一系列的改进和调整 将使用两个目标中精心制作的指标进行评估。满足这些指标将触发承诺， TrakITNow支持第三阶段的资金，以推出一个组合产品， 目标1将开发和衡量火鸟的化学OA 1架构，将转换Moskeet从一个 将捕获-计数-物种-通信产物转化为捕获-计数-物种-样品-通信产物。这将 利用Firebird的第一阶段创新技术（SterileQTM），自动捕获和稳定病毒RNA。不同 诱饵，RNA稳定防腐剂和预扩增酶将被计量，以创建，在年底， 第一年，一种Moskeet就绪的OA 1化学品，允许对蚊子进行连续采样。包括（a） >50%的感染蚊子留下可检测的样品，（B）>200小时的RNA半衰期，和（c）>1小时的捕获 pg的RNA。来自Insilixa（Hydra）的仪器，也通过Firebird的试剂增强，将用于现场 病毒检测Hydra是便携式的，其低复杂性的设计允许业余爱好者使用。 目标2将开发和衡量火鸟的化学OA 2架构。这将把莫斯基特变成一个 捕获-计数-特殊-样品-分析-交流产品。在这里，我们将开发和基准点- 取样扩增工具，具体地：（a）RT-PCR，和（B）环扩增（LAMP）。这些将作为 结合试剂创新（AEGIS、SAMRS、Biversals），以支持高度多重检测 虫媒病毒，创建多种分析物的均匀扩增，管理引物-引物产物，防止 PCR资源，以脱靶过程，创造“怪物”，并管理虫媒病毒序列分歧。

Field Detection of arboviruses directly from mosquito traps Firebird Biomolecular Sciences, LLC. Abstract Mosquitoes are the world's deadliest creatures. In the US, mosquitoes are vectors for West Nile, many kinds of encephalitis, and viruses arriving Latin America and Africa. In the developing world, they cause millions of cases of disease and death each year. Routine insect spraying is not a "fix", as it leads to pesticide resistance, encounters public opposition, and destroys beneficial insects. Thus, the EPA, CDC, ECDPC, and their counterparts around the globe, strongly recommend surveillance before spraying. Florida law requires it. In Phase 1, we created (a) innovative technology to generate CO2 to attract mosquitoes, (b) innovative tech- nology to collect viral RNA from the attracted mosquitoes, (c) data showing that collected RNA is stable under Florida summer conditions for at least 3 days, (d) processes where captured viral RNA is amplified directly without handling mosquitos or Qiagen RNA preps, and (e) reagents that allow robust multiplex amplification. These specs, which can transform the science and practice of arbovirus surveillance, attracted TrakITNow, an innovator in instrument design. Its award-winning trap (Moskeet) counts and speciates mosquitoes, doing everything short of collecting samples from the mosquitoes and determining if these hold arboviruses. This Phase 2 project will re-configure and metric the Phase 1 innovations to allow them, in Phase 3, to be placed on Moskeet in two operational architectures (OA1 and OA2). A series of improvement and adaptations will be evaluated using tightly crafted metrics in two Aims. Meeting these metrica will trigger a commitment by TrakITNow to support Phase 3 funding to launch a combined product, Aim 1 will develop and metric Firebird's chemistry for an OA1 architecture that will convert Moskeet from a trap-count-speciate-communicate product into a trap-count-speciate-sample-communicate product. This will exploit Firebird's Phase 1 innovation (SterileQTM) that autonomously traps and stabilizes viral RNA. Different baits, RNA-stabilizing preservatives, and pre-amplification enzymes will be metricked to create, at the end of Year 1, a Moskeet-ready OA1 chemistry that allows continuous sampling of mosquitoes. Metrics include (a) >50% of infected mosquitoes leaving detectable sample, (b) >200 hours half-life of RNA, and (c) capture of >1 pg of RNA. An instrument from Insilixa (Hydra), also enhanced by Firebird's reagents, will be used for on-site virus detection. Hydra is portable, and its low complexity design allows it to be used by amateurs. Aim 2 will develop and metric Firebird's chemistry for an OA2 architecture. This will convert Moskeet into a trap-count-speciate-sample-analyze-communicate product. Here, we will develop and benchmark point-of- sampling amplification tools, specifically: (a) RT-PCR, and (b) loop amplification (LAMP). These will, as needed, incorporate reagent innovations (AEGIS, SAMRS, Biversals) to support highly multiplexed detection of arboviruses, create uniform amplification of multiple analytes, manage primer-primer products, prevent loss of PCR resources to off-target processes that create "monsters", and manage arbovirus sequence divergence.