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

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纯化和分析核酸的工具的发展。目前研究RNA事务的过程，如转录、翻译和加工（例如，北方印迹、RNA微阵列、RNA测序和逆转录RT-PCR分析）可能是复杂和耗时的，并且通常需要专门的仪器或受过训练的人员。这些程序需要具有特定温度储存要求的试剂，并且是基于实验室的，这限制了它们用于POC测试的便携性和实用性，特别是在资源贫乏的环境中。为了便于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，很少有系统成功地整合了所有这些步骤。由于集成样品制备的复杂性，它在很大程度上被从这些系统中省略，并留给台式设备。该项目的总体目标是开发一种新的过程，可以进行完整的，自动化的，样本到答案的RNA检测分析，并提供使用RNA病毒的感染的准确的护理点（POC）诊断。在我们最初的第一阶段，可行性研究将集中在基因组登革热RNA的纯化和检测。我们将介绍一种新的自动化方法，用于从多种生物体（SPM）中纯化病毒基因组RNA，并在Lynntech的对流扩增装置中使用低功率RT-PCR技术分析RNA。这种方法将允许诊断登革热感染的发热期并区分登革热的血清型。该过程需要最少的培训，并提供了一个样本回答系统，简化了操作，减轻了用户错误的风险，并减少了整体检测时间。此外，该过程不依赖于实验室，因此有利于由非专业人员在“现场”进行RNA研究。拟议的过程将使科学家能够纯化RNA，通过RT-PCR进行RT和分析序列，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）开发便携式RT-PCR测定法，以使用对流扩增来鉴定RNA;以及3）使样品制备装置自动化。第二阶段将专注于将样本到答案的RNA检测过程适应于实时POC检测系统。我们将SPM和对流扩增系统集成到一个便携式单元中，该单元可以超越传统实验室的限制，用于研究RNA过程，以及诊断登革热等感染。Lynntech的实时检测系统将成为下一代工具，用于将分子诊断交付给全球医疗保健专业人员。这一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。最终产品预计将是FDA批准的，CLIA豁免的基于RNA的样本到答案的诊断设备，基于第一阶段和第二阶段建立的过程。

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.