Nanotrap particle viral enrichment technology for enabling portal, next-generation sequencing-based surveillance

项目总结 RNA病毒，如流感和埃博拉病毒，已被归类为导致 鉴于它们的高突变率和在宿主物种之间跳跃的能力，这种病毒在全球范围内流行。国家和全球 各机构呼吁进行研究和开发工作，以提高RNA病毒的监测能力。而当 在过去的二十年里，用于监测DNA病毒的工具有了巨大的进步，其独特的特性 一些RNA病毒已经挫败了为它们开发类似监测工具的尝试。当前最先进的技术 RT-qPCR和基因组测序等筛查方法需要重型设备和湿法实验室设施， 这阻碍了联邦机构可现场部署的监视和监测做法的进展，如 作为疾病控制中心的流感基因组团队(IGT)。此外，病原体从 生物样本在感染过程中迅速腐烂，仅几天后就下降到可检测到的水平以下 症状出现。因此，需要定义用于RNA病毒样本浓缩的新的协议和设备 它集速度、成本效益、易用性和现场操作能力于一身。这一需求是 IGT的早期实地工作强调了这一点，它利用了一种潜在的可现场部署的新病毒监测 测序方法，但表明该方法缺乏敏感性和特异性。 IGT和申请者CERES Nanosciences之间的合作导致了目前的SBIR阶段 I倡议，旨在测试利用Ceres的创新纳米颗粒技术的可行性，称为 NanoTrap(NT)，作为一种配套的病毒浓缩设备，价格低廉，易于使用，并且足够快速地 在使用疾控中心和其他机构最近开发的RNA和DNA病毒测序工具之前进行整合 组织。现在的主要技术目标是将这项工作转化为可使用的便携式设备 从现场采集的样本中浓缩病毒，以便与现场部署配合使用 病毒测序工具。这项工作将通过以下具体目标在九个月内完成 项目期间：目标1将演示使用NT颗粒捕获和浓缩流感的可行性 来自低滴度(30ct；30ct)的病原体，人为的病毒传输介质(VTM)样本，并提高附属物的敏感性； AIM 2将测试在基因组之前使用启用NT的预富集步骤的性能特征 测序以检测呼吸道病原体的存在。 这项工作的重要性将是产生一种技术来显著提高 在样本中传播的已知和新的RNA或DNA病毒株的鉴定和特性 从野外收集的。这项工作产生的长期商业产品将是一个优化的， 经过验证的、可现场部署的病毒浓缩设备，将由CDC、美国农业部、国土安全部、 国防部和任何其他监测基于RNA或DNA的传染病的实验室。

PROJECT SUMMARY RNA viruses, such as influenza and the Ebola virus, have been classified as the greatest threat for causing a global pandemic given their high mutation rates and abilities to jump between host species. National and global agencies have called for research and development efforts to improve RNA virus surveillance capabilities. While the tools for surveilling DNA viruses have advanced dramatically over the last two decades, the unique properties of RNA viruses have thwarted attempts to develop similar surveillance tools for them. Current state-of-the-art screening methods, like RT-qPCR and genome sequencing, require heavy equipment and wet-lab facilities, which has hindered progress of field-deployable surveillance and monitoring practices by federal agencies, such as the Centers for Disease Control’s Influenza Genomics Team (IGT). Furthermore, pathogen loads from biological samples decay rapidly over an infection’s course, decreasing below detectable levels only days after symptoms appear. Thus, there is a need to define new protocols and devices for RNA virus sample enrichment that combine speed, cost-effectiveness, ease of use, and the capacity for field operation. This need is underscored by early field work by the IGT, which utilized a potential new field-deployable viral surveillance sequencing protocol but demonstrated that the sensitivity and specificity of this protocol is lacking. Collaborations between the IGT and the applicant, Ceres Nanosciences, has led to the current SBIR Phase I initiative, aimed at testing the feasibility of leveraging Ceres’ innovative nanoparticle technology, termed Nanotrap (NT), as a companion virus enrichment device that is inexpensive, easy-to-use, and rapid enough to integrate prior to the use of RNA and DNA viral sequencing tools recently developed by the CDC and other organizations. The major technical objective now is to transition this work into a portable device that can be used to enrich viruses from samples collected in the field, so that it can be used in coordination with field-deployable viral sequencing tools. This work will be accomplished through the following specific aims over a nine-month project period: Aim 1 will demonstrate the feasibility of using NT particles to capture and concentrate influenza pathogens from low-titer (<30 ct), contrived viral transport media (VTM) samples and improve MinION sensitivity; Aim 2 will test the performance characteristics of using the NT-enabled pre-enrichment step prior to genomic sequencing to detect the presence of respiratory pathogens. The importance of this work would be the generation of a technology to significantly improve the rapid identification and characterization of known and novel strains of RNA or DNA viruses circulating in samples collected from the field. The long-term commercial product resulting from this work would be an optimized, validated, field-deployable virus enrichment device that would be marketed for use by the CDC, USDA, DHS, DoD, and any other laboratories that monitor RNA- or DNA-based infectious diseases.