spFRET for Expression Profiling mRNA: Discovering Markers for Stroke Diagnosis

spFRET 用于 mRNA 表达谱分析：发现中风诊断标记物

• 批准号: 8228093
• 负责人: Alison E Baird
• 金额: $ 57.96万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8228093
• 关键词: Adult; Architecture; Automation; Biochemical Process; Biological Assay; Biological Markers; Blood; Blood Platelets; Brain hemorrhage; Cell Fraction; Cell Separation; Cells; Clinical; Clinical Data; Clinical Research; Clinical Sensitivity; Complementary DNA; Coupled; Cytolysis; Data Collection; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Dimensions; Enzymes; Erythrocytes; Event; Extravasation; Fiber Optics; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Hour; Housekeeping Gene; Image; Immunoassay; In Vitro; Ischemic Stroke; Ligase; Light; Liquid substance; Magnetic Resonance Imaging; Messenger RNA; Microfluidics; Molecular; Molecular Diagnostic Testing; Molecular Profiling; Monitor; Nucleic Acids; Optics; Output; Patients; Performance; Peripheral; Phase; Photons; Pilot Projects; Poly T; Polymers; Process; RNA marker; Reaction; Reaction Time; Reader; Reverse Transcription; Sampling; Secure; Sensitivity and Specificity; Series; Serum Proteins; Signal Transduction; Solid; Stroke; Symptoms; System; Technology; Testing; Therapeutic Intervention; Time; Validation; Whole Blood; X-Ray Computed Tomography; base; cost; design; digital; disability; flexibility; innovation; innovative technologies; kinematics; mRNA Expression; molecular marker; neutrophil; new technology; novel; novel marker; optical fiber; peripheral blood; prevent; prototype; public health relevance; seal; single molecule; submicron; tool

DESCRIPTION (provided by applicant): Stroke is the third leading killer in the US and the main cause for over 795,000 cases of adult disability each year. The two major types of stroke, ischemic and hemorrhagic, cannot be clinically differentiated; 30% of patients presenting stroke-like symptoms do not have a stroke at all. It is imperative that a stroke diagnosis be made quickly and accurately because ischemic and hemorrhagic strokes require different treatments that have only a small window of time to be effective (3-6 hours). Computed tomography (CT) or magnetic resonance imaging (MRI) is commonly used for the diagnosis, which delays therapeutic intervention. Unfortunately, a molecular diagnostic test does not currently exist for stroke due primarily to the fact that potential molecular marker panels are waiting to be clinically validated. This validation process is, in part, hampered by the tools used for their analysis, such as reverse-transcription PCR (RT-PCR) for nucleic acid-based markers or immunoassays for serum protein markers, due to the lack of sample process automation and the lengthy processing steps used. In this application, a highly innovative, fully-automated system will be developed that has exquisite analytical sensitivity to monitor minute changes in the expression levels of different molecular markers with a turn-around-time (TAT) of less than 15 minutes. The system will facilitate the identification and validation of new molecular markers for the rapid, specific, and sensitive diagnosis of stroke. The molecular assay and novel hardware will be used to evaluate the potential of a messenger RNA marker panel as an unprecedented blood-based test (using peripheral blood mononucleated cells, PBMC) for the diagnosis of stroke. Two technologies will form the core of the system: (1) modular fluidic bio-processor made from polymers via replication that contains all of the sample processing steps; and (2) single pair fluorescence resonance energy transfer (spFRET) that eliminates several sample processing steps. Micro-replication will be used for the fabrication of the bio-processor to keep cost low and make this consumable appropriate for single- use applications, as demanded by clinical diagnostics. The bio-processor will contain a fluidic motherboard with task-specific modules interconnected on it to provide design flexibility. The molecular processing steps poised on the processor will be: (1) PBMC isolation; (2) PBMC lysis; (3) mRNA solid-phase purification; (4) reverse transcription to cDNA; (5) ligase detection reactions (LDRs) to generate molecular beacons with donor/acceptor fluorescent pairs and; (6) spFRET digital detection. The molecular (i.e., digital) counting using spFRET will provide the ability to eliminate several sample pre-processing steps shortening the assay TAT and generate the necessary analytical sensitivity to detect subtle changes in expression levels of mRNA markers, which may be critical during early times following a stroke event. The system can potentially be used for any application requiring mRNA expression profiling, but will be used in the current project for validating molecular markers for the diagnosis of stroke. PUBLIC HEALTH RELEVANCE: Stroke is the third leading killer in the US and causes over 795,000 cases of adult disability each year. Unfortunately, in vitro diagnostic tests for stroke that can be administered to patients for effective and timely treatment are not currently available. The goal of this project is to develop an innovative, discovery-based technology for securing highly quantitative results quickly through full-process automation. The technology will assist in the validation of different molecular markers for stroke to deliver an unprecedented in vitro diagnostic blood-based test.

描述（由申请人提供）：中风是美国的第三大杀手，也是每年有795,000例成人残疾案件的主要原因。两种主要类型的中风，缺血性和出血性，不能在临床上分化。 30％的患者出现中风症状根本没有中风。必须快速，准确地进行中风诊断，因为缺血性和出血性中风需要不同的治疗方法，而这些治疗只有很小的时间才能有效（3-6小时）。计算机断层扫描（CT）或磁共振成像（MRI）通常用于诊断，延迟治疗干预。不幸的是，由于潜在的分子标记面板正在等待临床验证，目前尚不存在中风的分子诊断测试。该验证过程部分受到用于分析的工具的阻碍，例如，由于缺乏样品过程自动化和使用的冗长处理步骤，用于基于核酸的标记的反向转录PCR（RT-PCR（RT-PCR）或血清蛋白标记的免疫测定）。在此应用程序中，将开发一个高度创新的，完全自动化的系统，具有精致的分析灵敏度，可监视不同分子标记的表达水平的微小变化，而转折时间（TAT）少于15分钟。该系统将促进和验证新分子标记，以快速，特定和敏感的中风诊断。分子测定和新型硬件将用于评估信使RNA标记面板作为前所未有的血液测试（使用外周血单核细胞，PBMC）的潜力，以诊断中风。两种技术将构成系统的核心：（1）由聚合物通过复制制成的模块化流体生物处理器，其中包含所有样品处理步骤； （2）单对荧光共振能量转移（SPFRET），它消除了几个样品处理步骤。根据临床诊断的要求，将使用微复制来制造生物处理器，以保持低成本，并使此易于使用，适合于单使用应用程序。生物处理器将包含一个流体主板，其中特定于任务的模块在其上互连以提供设计灵活性。在处理器上有序的分子处理步骤将是：（1）PBMC隔离； （2）PBMC裂解； （3）mRNA固相纯化； （4）向cDNA的逆转录； （5）连接酶检测反应（LDRS）以产生具有供体/受体荧光对的分子信标和； （6）SPFRET数字检测。使用SPFRET的分子计数（即数字）计数将提供消除几个样品预处理步骤缩短测定TAT并产生必要的分析灵敏度以检测mRNA标记表达水平的细微变化的能力，这在中风事件后的早期可能至关重要。该系统可能可能用于任何需要mRNA表达分析的应用，但将用于当前项目，以验证诊断中风的分子标记。 公共卫生相关性：中风是美国第三大杀手，每年导致超过795,000例成人残疾。不幸的是，目前尚无对患者进行有效和及时治疗的中风的体外诊断测试。该项目的目的是开发一种创新的，基于发现的技术，以通过完整的自动化快速获得高度定量的结果。该技术将有助于验证不同分子标记的中风，以提供前所未有的体外诊断基于血液的测试。