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

项目摘要 RNA病毒，如流感病毒和埃博拉病毒，已被列为最大的威胁，因为它会引起 由于它们的高突变率和在宿主物种之间跳跃的能力，它们在全球大流行。国家和全球 各机构呼吁进行研究和开发工作，以提高RNA病毒监测能力。而 在过去的二十年里，监控DNA病毒的工具有了巨大的进步， 对RNA病毒的研究阻碍了为它们开发类似监测工具的尝试。目前最先进的技术 筛选方法，如RT-qPCR和基因组测序，需要重型设备和湿实验室设施， 这阻碍了联邦机构实地部署监视和监测做法的进展， 作为疾病控制中心的流感基因组学小组（IGT）。此外，来自 生物样本在感染过程中迅速腐烂，仅在感染后几天就降至可检测水平以下。 症状出现。因此，需要定义用于RNA病毒样品富集的新方案和装置 该联合收割机结合了速度、成本效益、易用性和现场操作能力。这种需要 IGT的早期实地工作强调了这一点，IGT利用了一种潜在的新的可实地部署的病毒监测手段 测序方案，但证明该方案缺乏灵敏度和特异性。 IGT和申请人Ceres Nanosciences之间的合作导致了当前的SBIR阶段 该倡议旨在测试利用Ceres创新纳米颗粒技术的可行性， Nanotrap（NT）作为一种伴随病毒富集装置，价格低廉，易于使用，并且足够快速， 在使用CDC和其他机构最近开发的RNA和DNA病毒测序工具之前， 组织的现在的主要技术目标是将这项工作转变为可以使用的便携式设备 从实地收集的样本中富集病毒，以便与实地部署的 病毒测序工具。这项工作将在九个月内通过以下具体目标完成： 项目期间：目标1将证明使用NT颗粒捕获和浓缩流感的可行性 从低滴度（<30 ct）、人工病毒转运培养基（VTM）样本中分离病原体，并提高MinION灵敏度; 目的2将测试在基因组测序之前使用NT启用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，以显着提高快速 在样本中传播的已知和新的RNA或DNA病毒株的鉴定和表征 从外地收集的。这项工作产生的长期商业产品将是一种优化的， 经验证的、可现场部署的病毒富集装置，将销售给CDC、USDA、DHS使用， 美国国防部和任何其他监测RNA或DNA为基础的传染病的实验室。

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.