Point-of-Care RT-PCR System to Inform COVID-19 and Respiratory Illness Decisions

项目摘要 正在进行的新冠肺炎/SARS-CoV-2大流行突出表明，需要简单、快速和具有成本效益的 在护理地点进行呼吸道感染检测，包括医生办公室、紧急护理环境、 流动的程序中心和低资源环境。对于呼吸道来说，这种需求尤其值得注意 新冠肺炎和流感等感染，它们可以出现类似的症状，但需要不同的 管理策略。RT-pcr以其高度的敏感性和特异性成为检测分子水平的金标准。 呼吸道病原体的诊断和鉴别。传统的RT-PCR工作流程需要大量 对样本含量和反应条件的控制，目前的方法需要提取核酸 在扩增和检测之前。最终结果是增加了复杂性、成本和/或周转时间 诊断。在这方面，我们在最近流感研究中观察到，出色的分析表现 通过应用我们的新工作流程和自适应PCR，可以在不提取RNA的情况下获得特征 技术与传统的逆转录聚合酶链式反应不同，适应性逆转录聚合酶链合成了镜像的L-脱氧核糖核酸对映体- 在序列上与PCR引物和靶标相同-修改循环条件以匹配生化 样品含量，从而消除了监控反应温度的需要。对反应的直接监控 这些条件克服了传统聚合酶链式反应的许多局限性，便于在原始基因中直接扩增。 样品矩阵，简化了仪器设计，实现了单管分析。 SARS-CoV-2和流感都是有包膜的RNA病毒，样本收集在同一 方法(即鼻咽拭子)和使用相同的病毒传输介质。因此，我们假设 我们可以通过执行适应性RT-PCR来消除这种病毒的RNA提取，就像我们对流感所做的那样 直接在临床标本上。我们建议通过自适应RT-PCR实现简化的方法， 无需提取核糖核酸即可诊断新冠肺炎和其他呼吸道病原体。作为协作 在生物医学工程师和新冠肺炎诊断实验室之间，我们寻求开发一种工作流程和 简单易用、经济实惠且适用于护理点设置的工具，这些工具可以快速 告知治疗和管理策略。为了实现这一目标，目标1将评估RT- 使用传统和自适应RT-PCR仪器直接--也就是不提取RNA--进行聚合酶链式反应。 AIM 2将开发多重扩增试剂，以创建敏感和特异的呼吸面板 检测SARS-CoV-2、其他四种病毒、两种细菌和一个控制目标。最终，Aim 3将设计和 制作一种适用于护理点设置的自含式自适应RT-PCR仪器，同时验证这一点 在CLIA认可的实验室环境中使用特性化的人体样本的系统。本项目竣工 将为已有的和新出现的呼吸道感染带来一种新的护理点工具，这些感染威胁到 公共卫生，促进快速治疗、后续行动、感染预防和流行病控制。

Project Summary The ongoing COVID-19/SARS-CoV-2 pandemic highlights the need for simple, rapid, and cost-effective testing for respiratory infections at the point-of-care, including physician’s offices, urgent-care settings, ambulatory procedural centers, and low-resource environments. The need is particularly notable for respiratory infections, such as COVID-19 and influenza, which can present with similar symptoms yet require distinct management strategies. With its high sensitivity and specificity, RT-PCR is the gold standard for the molecular diagnosis and differentiation among respiratory pathogens. Traditional RT-PCR workflow requires significant control over specimen contents and reaction conditions, with current methods requiring nucleic acid extraction prior to amplification and detection. The net result is increased complexity, cost, and/or turnaround time for diagnosis. In this context, we have observed in recent influenza studies that outstanding analytic performance characteristics can be achieved without RNA extraction, by applying our novel workflow and Adaptive PCR technology. Unlike traditional RT-PCR, Adaptive RT-PCR incorporates mirror-image L-DNA enantiomers— identical in sequence to PCR primers and targets—that modify cycling conditions to match the biochemical sample contents, thus eliminating the need to monitor reaction temperature. The direct monitoring of reaction conditions overcomes many of the limitations of traditional PCR, facilitating direct amplification within the original specimen matrix, simplifying instrument design, and enabling single-tube analyses. SARS-CoV-2 and influenza are both enveloped RNA viruses, with specimens collected in the same manner (i.e. nasopharyngeal swab) and using the same viral transport medium. Therefore, we hypothesize that we may eliminate RNA extraction for this virus, like we have done for influenza, by performing Adaptive RT-PCR directly on clinical specimens. We propose to enable a simplified methodology through Adaptive RT-PCR, creating diagnostics for COVID-19 and other respiratory pathogens without RNA extraction. As a collaboration between biomedical engineers and a COVID-19 diagnostic laboratory, we seek to develop a workflow and instrument that are simple-to-use, cost-effective, and suitable for point-of-care settings, tools that can rapidly inform treatment and management strategies. To achieve this goal, Aim 1 will evaluate the performance of RT- PCR directly – that is, without RNA extraction – using both traditional and Adaptive RT-PCR instrumentation. Aim 2 will develop multiplexed amplification reagents to create a sensitive and specific respiratory panel that detects SARS-CoV-2, four other viruses, two bacteria, and one control target. Ultimately, Aim 3 will design and fabricate a self-contained Adaptive RT-PCR instrument suitable for point-of-care settings, while validating this system using characterized human specimens in a CLIA-accredited lab environment. Completion of this project will result in a novel point-of-care tool for both the established and emerging respiratory infections that threaten public health, facilitating rapid treatment, follow-up, infection prevention, and epidemiologic containment.