Microfluidic platform for rapid influenza subtyping

用于快速流感亚型分析的微流控平台

基本信息

批准号：
7847511
负责人：
ANUBHAV TRIPATHI
金额：
$ 18.72万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-22 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7847511
关键词：
Address Antiviral Agents Area Asia Avian Influenza Beds Binding Biomedical Engineering Cessation of life Chemicals Chemistry Clinical Code Communicable Diseases Complementary DNA Complex Consensus Sequence Cues DNA DNA Primers DNA Sequence Data Databases Detection Devices Diagnosis Diagnostic tests Environment Enzymes Epidemiology Fluorescence Fluorescent Probes Genes Genetic Transcription Glass Glean Goals Health Health Professional Healthcare Hemagglutinin Human Human Virus Immunity In Vitro Individual Infection Influenza Influenza A Virus, H1N1 Subtype Influenza A Virus, H5N1 Subtype Institution Label Laboratories Lasers Left Life Link Measures Messenger RNA Methods Microfluidic Microchips Microfluidics Microscope Microspheres Modeling Molecular Molecular Biology Techniques Monitor Nasal Lavage Fluid Neuraminidase Noise Oligonucleotides Outcome Pathogenicity Performance Persons Physicians Polymerase Population Population Surveillance Preparation Principal Investigator Production Property Public Health Quartz RNA RNA Sequences RNA Viruses RNA amplification RNA chemical synthesis Reaction Reaction Time Records Recurrence Research Reverse Transcription Sampling Self-Sustained Sequence Replication Signal Transduction Solid Solutions Specificity Specimen Speed Stream Streptavidin Structural Genes Surface Swab System T7 RNA polymerase Techniques Thermodynamics Time United States Upper respiratory tract Vaccines Variant Viral Viral Drug Resistance Viral Load result Viral Proteins Virus Work base biosafety level 2 facility clinical material cost density design detector experience fluorophore genetic analysis hazard hospital laboratories human disease influenza outbreak influenzavirus internal control microchip nanolitre novel nuclease pandemic disease pandemic influenza pathogen point of care pre-clinical pressure programs promoter quantum resistance mutation response solid state success transmission process viral RNA

项目摘要

Program Director/Principal Investigator (Last, First, Middle): ANUBHAV TRIPATHI, 1 R21 AI 73808-01A1 In this collaborative program, we will develop an integrated Influenza Detection Microchip capable of performing identification of sequence-specific influenza subtypes (H1, H3, and H5). Our device accepts clinical specimens from mucosal swabs and isolates influenza virus RNA (vRNA) based on conserved consensus sequences to complementary DNA oligonucleotides immobilized on microbeads. These sequences are used as templates for reverse transcription utilizing Nucleic Acid Sequence Based Amplification (NASBA) mechanism inside the chamber of a packed bed microchip. The transcription products of the continuous flow reaction are sequentially mixed with flow stream containing dual probes (also called molecular beacons) specific for one of each of the repertoire of possible H sequences. The dual probes fluoresce only when specifically hybridized with the complementary viral sequence. A low cost camera records the down stream detection area to collect the transient signal to identify the viral subtype; the results are then compared to the positive and negative controls. The proposed program will significantly advance fundamental understanding of physical & chemical cues necessary in an isothermal and exponential continuous flow amplification and detection of vRNA microreactor. The proposed device has significant advantages over current methods of detection and genetic analysis of infections outside of core lab facilities. The main advantages include rapid identification, high sensitivity, accuracy, minimal. sample preparation time and high information content. The combination of this experienced team of infectious disease physicians (Artenstein and Opal) and microfluidic bioengineers (Tripathi, IVIcCalia & Sarma) provides a unique advantage to completion of this goal. This research team has extensive experience with microfluidics, infectious diseases and advanced molecular biology techniques, which are all critical to the success of this project. The outcome of the program has a potential to provide public health laboratories, hospital clinical laboratories, and point-of-care facilities for highly sensitive and specific influenza diagnostic tests that rapidly generate a virologic diagnosis, subtype-specific information and epidemiologic tracking of influenza.

项目总监/首席研究员（姓、名、中）：ANUBHAV TRIPATHI, 1 R21 AI 73808-01A1 在这个合作项目中，我们将开发一种集成的流感检测微芯片，能够识别序列特异性流感亚型（H1、H3 和 H5）。我们的设备接受来自粘膜拭子的临床样本，并根据固定在微珠上的互补 DNA 寡核苷酸的保守共有序列分离流感病毒 RNA (vRNA)。这些序列用作逆转录的模板，利用填充床微芯片室内的基于核酸序列的扩增 (NASBA) 机制。连续流动反应的转录产物依次与含有双探针（也称为分子信标）的流动流混合，该双探针对每种可能的 H 序列库具有特异性。双探针仅在与互补病毒序列特异性杂交时才会发出荧光。低成本摄像机记录下游检测区域以收集瞬态信号以识别病毒亚型；然后将结果与阳性和阴性对照进行比较。拟议的计划将显着促进对 vRNA 微反应器的等温和指数连续流动扩增和检测所需的物理和化学线索的基本理解。与当前核心实验室设施之外的感染检测和遗传分析方法相比，所提出的设备具有显着优势。主要优点包括识别快速、灵敏度高、准确度低。样品制备时间短且信息含量高。这支经验丰富的传染病医生团队（Artenstein 和 Opal）与微流体生物工程师（Tripathi、IVicCalia 和 Sarma）的结合为实现这一目标提供了独特的优势。该研究团队在微流控、传染病和先进分子生物学技术方面拥有丰富的经验，这些对于该项目的成功至关重要。该计划的成果有可能为公共卫生实验室、医院临床实验室和护理点设施提供高度敏感和特异性的流感诊断测试，从而快速生成流感的病毒学诊断、亚型特异性信息和流行病学追踪。