Nucleic Acid Dipstick for Rapid Avian Flu Detection at Point-of-care

描述(申请人提供)：在过去的十年里，基于核酸的分子检测已经成为一种强大的疾病诊断工具，因为它比传统的免疫学方法具有更高的灵敏度和特异性。然而，这样的分子分析还不能用于POC临床，主要是因为与当前的核酸检测方法相关的复杂的设施和仪器要求，例如在核酸提取中的真空或离心法，以及在称为聚合酶链式反应(PCR)的核酸扩增过程中昂贵而笨重的热循环仪。为了开发用于POC和现场应用的下一代分子诊断工具，我们建议开发廉价(每个约10美元)、手持、易于操作的核酸试纸设备(圆珠笔大小，如商店出售的家用验孕试纸)，能够在60分钟内检测和区分多种流感样症状病原体(例如~H5N1禽流感、A型人类流感、RSV、SARS、腺病毒、副流感等)。我们试纸条检测/设备的典型设计包括：从临床样本(例如鼻拭子)中提取核酸的简单机械试剂盒；快速等温扩增疾病基因靶标序列；使用简单的比色标记(例如染色微球)标记扩增的疾病靶标序列；以及将标记的疾病基因序列捕获到基于夹心杂交的横向流动试纸棒上用于可视化。我们建议的具体目标是开发(1)简单的机械式核酸提取盒，以消除提取核酸所需的离心机(或真空)，(2)新型的等温核酸流感分析，以消除这种热循环，(3)手持侧向流动色谱试纸设备，可对扩增产物进行多路检测/可视化，以及(4)集成野战流感检测设备，能够在一个简单的手持试纸设备中同时检测/区分甲型流感、禽流感H5N1和其他几种类似流感的病毒：SARS、RSV、副流感。一旦开发出来，我们将通过与标准RT-PCR和培养分析进行并列比较，在临床样本上验证试纸条法。这种DMA DipStick设备的主要用途将是在没有复杂的研究设施和电力供应的现场进行医疗诊断和传染病筛查(例如禽流感和SARS)。这样的核酸试纸将提供前所未有的速度(现场结果)、特异性(基于基因的检测)、灵敏度(100个疾病细胞)、可负担性(10美元)和信息密度(一个简单的设备可以查询10种不同的疾病)。

DESCRIPTION (provided by applicant): In the past decade, nucleic acid-based molecular detection has become a powerful disease diagnosis tool due to its superior sensitivity and specificity over conventional immunological methods. However, such molecular assays have not been available for point-of-care (POC) clinics mainly due to elaborate facility and instrumentation requirements associated with current nucleic acid detection methods, e.g. vacuum or centrifugation in nucleic acid extractions, and an expensive and heavy thermal cycler apparatus in nucleic acid amplification procedure called polymerase chain reaction (PCR). To develop the next generation molecular diagnostic tools for POC and field applications, we propose to develop inexpensive (~$10 each), hand-held, easy-to-operate, nucleic acid dipstick devices (ball pen sizes, like those home pregnancy test strip sold in stores) capable of detecting and distinguishing multiple flu-like symptom pathogens (e.g. ~H5N1 avian flu, Type A human flu, RSV, SARS, adenoviruses, parainfluenza etc) in <60 minutes. A typical design of our dipstick assay/device involves the following: a simple mechanical cartridge extraction of nucleic acid from a clinical sample (e.g. nasal swab); rapid isothermal amplification of disease gene target sequences; labeling of the amplified disease target sequences using simple colorimetric labels (e.g. dyed microsphere); and capture the labeled disease gene sequences onto a sandwich hybridization-based lateral flow dipstick for visualization. The specific aims of our proposal is to develop (1) a simple mechanical nucleic acid extraction cartridge to eliminate centrifugation (or vacuum) requirement for nucleic acid extraction, (2) a novel isothermal nucleic acid influenza assay to eliminate such thermal cycling, (3) a hand-held lateral flow chromatography dipstick device allowing multiplexed detection/visualization of amplification products, and (4) an integrated field flu detection device capable of simultaneous detection/distinction of Influenza A, Avian Influenza H5N1, and several other flu-like viruses SARS, RSV, parainfluenza in one simple hand-held dipstick device. Once developed, we will validate dipstick assay on clinical samples by performing side-by- side comparison with standard RT-PCR and culturing assays. The primary use of such a DMA dipstick device will be point-of-care medical diagnosis and infectious disease screening (e.g. avian flu and SARS) in field where elaborate research facility and electricity is not available. Such a nucleic acid dipstick will provide unprecedented speed (on-site results), specificity (gene-based detection), sensitivity (<100 disease cells), affordability (<$10), and information density (>10 different diseases can be queried in one simple device).