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开发了非常强大的工具来净化和分析核酸。当前研究RNA交易的过程，例如转录，翻译和处理（例如，北印迹，RNA微阵列，RNA测序和逆转录RT-PCR分析）可能是复杂且耗时的，通常需要专业的仪器或训练有素的人员。这些程序需要具有特定温度的存储要求并且是基于实验室的试剂，这限制了其可移植性和用于POC测试的实用性，尤其是在资源贫乏的设置中。为了促进POC分析，将样品制备，样品处理，放大（PCR； RT-PCR）整合到一个自动化过程中至关重要。但是，很少有系统成功整合了所有这些步骤。由于整合样品准备的复杂性，因此从这些系统中省略了它，并剩下到台式设备。该项目的总体目标是开发一个可以执行完整，自动化的，样品到索引的RNA检测分析的新过程，并使用RNA病毒对感染的准确诊断（POC）诊断。在首次I阶段，可行性研究将集中于基因组登革热RNA的纯化和检测。我们将引入一种新型的自动化方法，用于从多个生物体（SPM）纯化病毒基因组RNA，并在Lynntech的对流放大单元中使用低功率RT-PCR技术分析RNA。该方法将允许诊断登革热感染的高热阶段，并在登革热的血清型之间进行区分。该过程需要最小的培训，并提供了一个样本到答案系统，该系统简化了操作，减轻了用户错误的风险并减少了总体检测时间。此外，该过程与实验室无关，因此有利于非专业人员在“现场”进行RNA研究。提出的过程将使科学家能够通过RT-PCR纯化RNA，执行RT和分析序列，并适用于转录分析，RNA检测和POC诊断。 I阶段的目的包括：1）开发便携式RNA样品制备模块； 2）开发一种便携式RT-PCR分析，以使用对流流扩增识别RNA； 3）自动化样品准备设备。第二阶段将着重于将样品之间的RNA检测过程调整为实时POC检测系统。我们将将SPM和对流放大系统整合到一个便携式单元中，该单元可以超出传统实验室的范围来研究RNA过程，并诊断登革热等感染。 Lynntech的实时检测系统将成为下一代工具，用于将分子诊断物交付给全球卫生保健专业人员手中。该系统将为无法提供传统测试的社区和国家提供必要的资源。根据I和II阶段中确定的过程，最终产品被设想为基于FDA批准的，基于CLIA的基于RNA的样品诊断设备。