Strain Specific Detection of Influenza at the Point-of-Care

在护理点对流感病毒株进行特异性检测

• 批准号: 7917202
• 负责人: HYONGSOK Tom SOH
• 金额: $ 37.8万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-18 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917202
• 关键词: Affinity; Aptamer Technology; Arts; Binding; Biochemistry; Capsid Proteins; Cessation of life; Chemistry; Complex; Coupling; DNA; DNA Microarray Chip; Detection; Development; Devices; Diagnostic; Dose; Engineering; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Food Safety; Generations; Genetic; Hour; Human; Influenza; Influenza A Virus, H5N1 Subtype; Influenza A virus; Joints; Label; Laboratories; Magnetism; Methods; Microfabrication; Microfluidic Microchips; Microfluidics; Monoclonal Antibodies; Morbidity - disease rate; Mucous body substance; Mutate; Mutation; Nose; Nucleoproteins; Output; Patients; Phase; Polymerase Chain Reaction; Preparation; RNA; RNA Sequences; Reading; Reagent; Recovery; Reporting; Reverse Transcription; Sampling; Security; Sensitivity and Specificity; Signal Transduction; Single-Stranded DNA; Solutions; Surface; Swab; System; Systems Analysis; Technology; Testing; The Sun; Time; Viral; Virus; Work; World Health Organization; aptamer; base; genetic analysis; influenzavirus; magnetic beads; miniaturize; mortality; novel; operation; pandemic disease; particle; pathogen; point of care; point-of-care diagnostics; protein complex; public health relevance; sensor; skills; success; viral detection; virology

DESCRIPTION (provided by applicant): Strain Specific Detection of Influenza at the Point-of-Care 1. PROJECT SUMMARY Influenza viruses cause significant morbidity and mortality worldwide; World Health Organization (WHO) estimates 250,000 ~ 500,000 yearly deaths. In particular, a highly pathogenic strain - avian influenza virus H5N1 (15, 56) - poses a threat of a possible pandemic because it can rapidly mutate to acquire the ability to transmit among humans (23, 49). The detection of minimal human infectious dose (~100 viral particles) (65) of Influenza A from patient samples (e.g. nasal swabs or mucus) is a significant analytical challenge. Detection by viral culture is sensitive and specific; however, this method is slow (3-10 days) and cannot be performed at POC. Surface marker-based detection (e.g. ELISA) provide a simple and rapid testing, however, it suffers from a poor limit of detection (~ 105 viral particles/ml) (9, 21, 59). Currently, polymerase chain reaction (PCR) offers the highest sensitivity in a much shorter turn-around time (2-4 hours) (29, 61, 67). Nevertheless, due to the difficulties in POC operation, patient samples are currently analyzed at central laboratories which typically requires ~2-3 days. Currently in the field, there is no automated technology solution that can perform specific influenza detection directly from patient samples with high sensitivity and specificity, and the WHO has specifically emphasized the critical need to fill this important technological gap (75). Towards a solution to this important problem, this group of three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop a powerful, field- portable, microfluidic platform for Influenza detection at POC. This effort is truly unique in many facets; first, to circumvent the problem posed by rapid mutation of the coat protein, we propose to generate novel DNA aptamer reagents which will specifically bind to the nucleoprotein complex of the virus. Currently, there are no reported aptamers for the nucleoprotein complex, and due to the fact that the nucleoprotein is conserved, it will serve as a universal tag to label Influenza A. Secondly, the aptamer reagent will be conjugated to magnetic beads to purify the nucleoprotein complex from nasal swab samples using our unique micromagnetics technology. Thirdly, by leveraging our expertise in miniaturized genetic analysis systems, we will develop the IMED chip capable of 1) integrated micromagnetic sample purification, 2) reverse transcription-PCR amplification and 3) sequence-specific electrochemical detection in a single monolithic device. The successful completion of this project will allow a quantitative "sample-to-answer" capability - the input into the system will be unprocessed patient samples, and the output will be an electrochemical signal that will be directly proportional to the number of copies of specific influenza RNA sequences in the sample. Such powerful combination of novel affinity reagents with highly integrated disposable devices will enable the development of critically needed effective POC diagnostics systems. PUBLIC HEALTH RELEVANCE: Strain Specific Detection of Influenza at the Point-of-Care Due to the fact that the envelope of influenza A virus differs among subtypes and evolves continuously, there is an urgent need for a field-portable genetic detection platform that can rapidly identify pathogenic strain of viral pathogens from unprocessed samples with high sensitivity and specificity. Towards an effective solution, the three PI's with complementary skill sets (influenza virology, aptamer biochemistry and microfluidic device engineering) propose to develop an effective point-of-care diagnostic system for influenza by 1) generating specific, high affinity DNA aptamers to tag the conserved regions of Influenza A, and 2) developing a highly integrated microfluidic system capable of magnetic sample preparation, RT-PCR amplification and sequence specific electrochemical detection in a single chip. By integrating the functions in a monolithic chip, the success of this project will yield a novel POC analysis system with unprecedented capabilities which will have a significant impact for Influenza A detection as well as many other applications in food safety, environmental monitoring and homeland security. 1

描述(由申请人提供)：在护理地点进行流感病毒株特异性检测1.项目摘要流感病毒在全球范围内造成显著的发病率和死亡率；世界卫生组织(WHO)估计每年有250,000至500,000人死亡。特别是，高致病性禽流感病毒H5N1(15，56)株对可能的大流行构成威胁，因为它可以迅速变异，获得在人类之间传播的能力(23，49)。从患者样本(例如鼻拭子或粘液)中检测最小人类感染剂量(~100个病毒颗粒)(65)是一项重大的分析挑战。通过病毒培养进行检测是敏感和特异的；然而，这种方法速度很慢(3-10天)，不能在POC进行。基于表面标志物的检测(例如，ELISA)提供了一种简单、快速的检测方法，但其检测限较低(~105个病毒颗粒/毫升)(9、21、59)。目前，聚合酶链式反应(PCR)在更短的周转时间(2-4小时)(29、61、67)内提供了最高的灵敏度。然而，由于POC操作的困难，目前患者样本在中心实验室进行分析，通常需要2-3天。目前在这一领域，没有一种自动化的技术解决方案可以直接从患者样本中进行高灵敏度和高特异性的特定流感检测，世卫组织特别强调了填补这一重要技术空白的迫切需要(75)。为了解决这一重要问题，这组拥有互补技能(流感病毒学、适体生物化学和微流控设备工程)的三名PI提议在POC开发一个强大的、现场便携的微流控平台来检测流感。这项工作在许多方面都是独一无二的；首先，为了绕过外壳蛋白快速突变带来的问题，我们建议开发新的DNA适体试剂，它将与病毒的核蛋白复合体特异结合。目前，核蛋白复合体的适体还没有报道，由于核蛋白的保守性，它将作为标记甲型流感的通用标签。其次，适体试剂将偶联到磁珠上，利用我们独特的微磁学技术从鼻拭子样本中纯化核蛋白复合体。第三，通过利用我们在小型化基因分析系统方面的专业知识，我们将开发能够在单个单片设备中实现1)集成微磁样品纯化、2)反转录-PCR扩增和3)序列特定电化学检测的IMED芯片。该项目的成功完成将实现定量的“样本对答案”能力--输入到系统中的将是未经处理的患者样本，输出将是与样本中特定流感RNA序列的拷贝数成正比的电化学信号。这种新型亲和试剂与高度集成的一次性设备的强大组合将使迫切需要的高效POC诊断系统的开发成为可能。公共卫生相关性：护理地点的流感病毒株特异性检测由于甲型流感病毒的包膜因亚型不同而不断进化，迫切需要一种现场便携的基因检测平台，能够从未经处理的样本中以高灵敏度和特异性快速识别病毒病原体的致病株。为了找到有效的解决方案，三个具有互补技能的PI(流感病毒学、适体生物化学和微流控设备工程)建议通过以下方式开发一种有效的临床点诊断系统：1)产生特定的、高亲和力的DNA适体来标记甲型流感的保守区；2)开发一种高度集成的微流控系统，能够在单个芯片中进行磁性样品制备、RT-PCR扩增和序列特异性电化学检测。通过将这些功能集成到一个单片芯片中，该项目的成功将产生一个具有前所未有的能力的新型POC分析系统，这将对甲型流感检测以及食品安全、环境监测和国土安全的许多其他应用产生重大影响。1