One-Step, POC Sample-to-Answer Process for RNA Analysis Outside the Laboratory

用于实验室外 RNA 分析的一步式 POC 样本到答案流程

• 批准号: 8523267
• 负责人: JOHN E MUELLER
• 金额: $ 22.16万
• 依托单位: LYNNTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2015-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8523267
• 关键词: Bedside Testings; Biological Assay; Blood; Buffers; Communicable Diseases; Communities; Complementary DNA; Complex; Country; Cytolysis; Data; Dengue; Detection; Development; Devices; Diagnosis; Diagnostic; Early Diagnosis; Early treatment; Equipment; FDA approved; Feasibility Studies; Fever; Gene Expression; Genetic Transcription; Genomics; Goals; Hand; Health; Health Professional; Human; Human Resources; Infection; Laboratories; Lead; Left; Life; Methods; Molecular; Northern Blotting; Nucleic Acids; One-Step dentin bonding system; Organism; Outcome; Phase; Preparation; Procedures; Process; RNA; RNA Processing; RNA Sequences; RNA Viruses; RNA analysis; RNA purification; Reaction; Reagent; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Risk; Sampling; Scientist; Sequence Analysis; Serotyping; Signal Transduction; Small Business Innovation Research Grant; System; Techniques; Technology; Temperature; Testing; Time; Training; Translation Process; Viral; Virion; Virus; Work; base; global health; improved; instrumentation; next generation; novel; operation; point of care; point-of-care diagnostics; portability; prototype; public health relevance; success; tool

DESCRIPTION (provided by applicant): Advances in molecular technologies over the past few decades have led to the development of very powerful tools to purify and analyze nucleic acids. Current processes to study RNA transactions such as transcription, translation and processing (e.g., Northern blot, RNA microarray, RNA sequencing and Reverse Transcription RT-PCR analysis) can be complex and time-consuming, and often require specialized instrumentation or trained personnel. These procedures require reagents that have temperature-specific storage requirements and are laboratory-based, which limit their portability and utility for POC testing, especially in resource-poor settings. To facilitate POC analysis, it i critical to integrate sample preparation, sample handling, amplification (PCR; RT-PCR) into a single, automated process. However, few systems have successfully integrated all of these steps. Due to the complexity of integrating sample prep, it has largely been omitted from these systems and left to bench-top equipment. The overall goal of this project is to develop a novel process that can perform a complete, automated, sample-to-answer RNA detection analysis and provide an accurate point-of-care (POC) diagnosis of infection using an RNA virus. During our initial Phase I, feasibility studies will focus on the purification and detection of genomic dengue RNA. We will introduce a novel automated method for purifying viral genomic RNA from multiple organisms (SPM) and analyzing that RNA using low-power RT-PCR techniques in Lynntech's convective amplification unit. This method will allow the diagnosis of the febrile phase of dengue infection and differentiate among serotypes of dengue. The process requires minimal training and provides a sample-to-answer system that simplifies operation, mitigates risk of user error, and decreases overall time-to-detection. Furthermore, the process is not tied to the laboratory, and therefore is conducive to performing RNA studies "in-the-field" by non-specialized personnel. The proposed process will enable scientists to purify RNA, perform RT and analyze sequences via RT-PCR and will be applicable to transcriptional analysis, RNA detection and POC diagnostics. Aims of the Phase I include: 1) develop a portable RNA sample preparation module; 2) develop a portable RT-PCR assay to identify RNA using convective flow amplification; and 3) automate the sample prep device. Phase II will focus on adapting the sample-to-answer RNA detection process to a real-time POC detection system. We will integrate SPM and convective amplification systems into a single portable unit that can be used beyond the confines of the traditional laboratory to study RNA processes, as well as to diagnose infections such as dengue. Lynntech's real-time detection system will be a next generation tool for delivering molecular diagnostics into the hands of health-care professionals globally. This system will provide necessary resources to communities and countries where traditional tests are not available. The final product is envisaged to be an FDA-approved, CLIA-waived RNA-based sample-to-answer diagnostic device based on the process established in Phase I and II.

描述（由申请人提供）：过去几十年来分子技术的进步导致了非常强大的核酸纯化和分析工具的开发。当前研究转录、翻译和加工（例如 Northern blot、RNA 微阵列、RNA 测序和逆转录 RT-PCR 分析）等 RNA 事务的过程可能非常复杂且耗时，并且通常需要专门的仪器或经过培训的人员。这些程序需要具有特定温度存储要求并且基于实验室的试剂，这限制了它们在 POC 测试中的便携性和实用性，特别是在资源匮乏的环境中。为了促进 POC 分析，将样品制备、样品处理、扩增（PCR；RT-PCR）集成到单个自动化过程中至关重要。然而，很少有系统能够成功集成所有这些步骤。由于集成样品制备的复杂性，这些系统在很大程度上省略了它，并留给了台式设备。该项目的总体目标是开发一种新颖的流程，可以执行完整、自动化、从样本到答案的 RNA 检测分析，并使用 RNA 病毒提供准确的即时护理 (POC) 感染诊断。在我们的第一阶段，可行性研究将集中于登革热基因组 RNA 的纯化和检测。我们将介绍一种新颖的自动化方法，用于从多种生物体 (SPM) 中纯化病毒基因组 RNA，并在 Lynntech 的对流放大装置中使用低功率 RT-PCR 技术分析该 RNA。该方法将允许诊断登革热感染的发热期并区分登革热血清型。该过程需要最少的培训，并提供一个样本到答案系统，可简化操作、降低用户错误风险并缩短总体检测时间。此外，该过程不依赖于实验室，因此有利于非专业人员“现场”进行RNA研究。拟议的流程将使科学家能够纯化 RNA、进行 RT 并通过 RT-PCR 分析序列，并将适用于转录分析、RNA 检测和 POC 诊断。第一阶段的目标包括：1）开发便携式RNA样品制备模块； 2) 开发一种便携式 RT-PCR 检测方法，利用对流扩增技术来识别 RNA； 3) 自动化样品制备装置。第二阶段将重点关注将样本到答案的 RNA 检测过程适应实时 POC 检测系统。我们将把 SPM 和对流放大系统集成到一个便携式装置中，该装置可以超越传统实验室的范围来研究 RNA 过程，以及诊断登革热等感染。 Lynntech 的实时检测系统将成为向全球医疗保健专业人员提供分子诊断的下一代工具。该系统将为无法进行传统测试的社区和国家提供必要的资源。最终产品预计将成为 FDA 批准的、CLIA 豁免的基于 RNA 的样本到答案诊断设备，基于第一阶段和第二阶段建立的流程。