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

描述（由申请人提供）：在过去的十年中，基于核酸的分子检测由于其比常规免疫学方法具有上级灵敏度和特异性而成为强大的疾病诊断工具。然而，这种分子测定法还不能用于护理点（POC）诊所，这主要是由于与当前核酸检测方法相关的复杂设施和仪器要求，例如核酸提取中的真空或离心，以及称为聚合酶链式反应（PCR）的核酸扩增程序中的昂贵且笨重的热循环仪装置。为了开发下一代POC和现场应用的分子诊断工具，我们建议开发廉价的（每个约10美元），手持式，易于操作，核酸试纸装置（圆珠笔大小，如商店出售的家用妊娠测试条），能够检测和区分多种流感样症状病原体（例如~ H5 N1禽流感、A型人流感、RSV、SARS、腺病毒、副流感等）。我们的试纸条测定/装置的典型设计涉及以下：从临床样品中提取核酸的简单机械盒（例如鼻拭子）;疾病基因靶序列的快速等温扩增;使用简单的比色标记物标记扩增的疾病靶序列（例如染色的微球）;和将标记的疾病基因序列捕获到基于夹心杂交的侧向流浸染条上用于可视化。我们建议的具体目标是开发（1）一种简单的机械核酸提取盒，以消除离心（或真空）要求，（2）消除这种热循环的新的等温核酸流感测定，（3）允许扩增产物的多重检测/可视化的手持侧流色谱试纸装置，和（4）能够在一个简单的手持试纸装置中同时检测/区分甲型流感、禽流感H5 N1和几种其它流感样病毒SARS、RSV、副流感的集成现场流感检测装置。一旦开发完成，我们将通过与标准RT-PCR和培养检测进行并行比较，验证试纸条检测在临床样本上的有效性。这种DMA试纸装置的主要用途将是现场护理医疗诊断和传染病筛查（例如禽流感和SARS），在现场，精密的研究设施和电力是不可用的。这种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（>10种不同的疾病可以在一个简单的设备中查询）。

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).