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

护理点 RT-PCR 系统可为 COVID-19 和呼吸道疾病决策提供信息

• 批准号: 10265605
• 负责人: Nicholas M Adams
• 金额: $ 78.88万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10265605
• 关键词: 2019-nCoV; Academic Medical Centers; Bacteria; Bacterial Infections; Biochemical; Biomedical Engineering; CLIA certified; COVID-19; COVID-19 assay; COVID-19 detection; COVID-19 diagnostic; COVID-19 pandemic; COVID-19 testing; Characteristics; Clinic; Clinical; Collaborations; Containment; DNA; Data; Device Designs; Devices; Diagnosis; Diagnostic; Engineering; Environment; Epidemiology; Goals; Gold; Health; Human; Image; Infection; Infection prevention; Infectious Diseases Research; Influenza; Laboratories; Light; Methodology; Methods; Molecular; Molecular Diagnosis; Monitor; Nucleic Acids; Optics; Patients; Performance; Physicians; Physicians' Offices; Population; Preparation; Public Health; RNA; RNA Viruses; RNase P; Reaction; Reagent; Reporting; Resources; Respiratory Tract Infections; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Sensitivity and Specificity; Specificity; Specimen; Supportive care; Symptoms; System; Technology; Temperature; Tennessee; Testing; Time; Training; Tube; Viral; Virus; Work; amplification detection; base; clinically relevant; cost; cost effective; design; diagnostic assay; enantiomer; experience; follow-up; instrument; instrumentation; molecular diagnostics; multiplex assay; nasopharyngeal swab; novel; pathogen; point of care; point-of-care diagnostics; preclinical evaluation; primary care setting; research clinical testing; respiratory; respiratory pathogen; tool; urgent care

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.

项目摘要 持续的COVID-19/SARS-CoV-2大流行凸显了对简单、快速和具有成本效益的 在护理点进行呼吸道感染检测，包括医生办公室，紧急护理环境， 流动手术中心和低资源环境。这一需求尤其值得注意， 感染，如COVID-19和流感，可能会出现类似的症状，但需要不同的 管理战略。RT-PCR具有高度的敏感性和特异性，是分子生物学检测的金标准。 呼吸道病原体的诊断和鉴别。传统的RT-PCR工作流程需要大量的 控制样本内容物和反应条件，当前方法需要核酸提取 在扩增和检测之前。最终的结果是增加的复杂性、成本和/或周转时间， 诊断.在这种情况下，我们在最近的流感研究中观察到， 通过应用我们的新工作流程和自适应PCR，无需RNA提取即可实现特征 技术.与传统的RT-PCR不同，自适应RT-PCR结合了镜像L-DNA对映体- 在序列上与PCR引物和靶相同-修改循环条件以匹配生物化学 样品内容物，从而无需监测反应温度。直接监测反应 条件克服了传统PCR的许多局限性，有利于在原始扩增条件下直接扩增。 样品矩阵，简化仪器设计，并使单管分析。 SARS-CoV-2和流感病毒都是有包膜的RNA病毒，标本收集在同一个 方法（即鼻咽拭子）和使用相同的病毒转运培养基。因此，我们假设 我们可以通过自适应RT-PCR来消除这种病毒的RNA提取，就像我们对流感病毒所做的那样， 直接应用于临床标本。我们建议通过自适应RT-PCR实现简化的方法， 为COVID-19和其他呼吸道病原体提供诊断，而无需提取RNA。以合作 在生物医学工程师和COVID-19诊断实验室之间，我们寻求开发一个工作流程， 使用简单、成本有效且适用于护理点设置仪器、可以快速 为治疗和管理战略提供信息。为了实现这一目标，目标1将评估RT的性能， 直接PCR-即不提取RNA-使用传统和自适应RT-PCR仪器。 Aim 2将开发多重扩增试剂，以创建一个敏感和特异的呼吸道面板， 检测SARS-CoV-2、其他四种病毒、两种细菌和一种对照靶标。最终，Aim 3将设计和 制造一个独立的自适应RT-PCR仪器，适用于护理点环境，同时验证这一点 在CLIA认证的实验室环境中使用特征化的人体标本的系统。完成本项目 将为现有和新出现的呼吸道感染提供一种新的即时护理工具， 公共卫生，促进快速治疗，后续行动，感染预防和流行病遏制。