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

现场直接从蚊子诱捕器中检测虫媒病毒 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，蚊子是西尼罗河的媒介，种类繁多 脑炎，以及抵达拉丁美洲和非洲的病毒。在发展中国家，它们造成数百万人 每年都有疾病和死亡病例。常规喷洒杀虫剂并不是解决办法，因为它会导致杀虫剂产生抗药性， 遭到公众的反对，并摧毁有益的昆虫。因此，EPA、CDC、ECDPC及其 全球同行强烈建议在喷洒前进行监测。佛罗里达州的法律要求如此。 在第一阶段，我们创造了(A)产生二氧化碳以吸引蚊子的创新技术，(B)创新技术- (C)数据显示，收集到的RNA在 佛罗里达州夏季条件至少3天，(D)直接扩增捕获的病毒RNA的过程 不需要处理蚊子或Qiagen RNA准备，以及(E)允许强大多重扩增的试剂。 这些规范可以改变虫媒病毒监测的科学和实践，吸引了TrakITNow和 仪器设计的创新者。它的获奖捕蚊器(Moskeet)对蚊子进行计数和分类， 除了从蚊子身上采集样本并确定这些蚊子是否携带虫媒病毒之外。 此阶段2项目将重新配置和衡量阶段1的创新，以使它们在阶段3 在两个业务架构(OA1和OA2)中放置在Moskeet上。一系列的改进和适应 将在两个目标中使用严密制定的指标进行评估。满足这些Metrica标准将触发以下承诺 TrakITNow支持第三阶段资金推出合并产品， 目标1将为OA1架构开发和度量Firebird的化学成分，该架构将把Moskeet从 陷阱-计数-指定-将产品传送到陷阱-计数-指定-样本-传送产品。这将是 利用Firebird的第一阶段创新(SterileQTM)自动捕获和稳定病毒RNA。不同 将对诱饵、稳定RNA的防腐剂和预扩增酶进行计量，以在 第一年，一种用于Moskeet的OA1化学物质，允许连续采集蚊子样本。衡量标准包括(A) 50%的受感染蚊子离开可检测的样本，(B)&gt；200小时RNA半衰期，以及(C)捕获&gt；1 Pg的核糖核酸。Insilixa(九头蛇)的一台仪器也得到了Firebird试剂的改进，将用于现场 病毒检测。九头蛇是便携的，其低复杂性的设计允许业余爱好者使用。 AIM 2将为OA2架构开发和度量Firebird的化学性能。这将把Moskeet转换为 Trap-count-speciate-sample-analyze-communicate产品。在这里，我们将开发和基准接入点 采样扩增工具，特别是：(A)RT-PCR和(B)环扩增(LAMP)。这些将作为 需要，结合试剂创新(宙斯盾、SAMRS、BUniversal)，以支持高度多元化的检测 虫媒病毒，创建多个分析物的统一扩增，管理底物-底物产品，防止丢失 聚合酶链式反应资源，以脱离目标的进程，创造“怪物”，并管理虫媒病毒的序列分歧。

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.