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

描述（申请人提供）：近十年来，基于核酸的分子检测因其优于常规免疫学方法的敏感性和特异性而成为一种强有力的疾病诊断工具。然而，这种分子测定法还不能用于即时护理（POC）诊所，主要原因是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，以及核酸扩增过程中称为聚合酶链反应（PCR）的昂贵且笨重的热循环仪。为了开发用于POC和现场应用的下一代分子诊断工具，我们建议开发价格低廉（每个约10美元），手持式，易于操作的核酸试纸设备（珠笔大小，如商店出售的家用妊娠试纸条），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型人流感，RSV， SARS，腺病毒，副流感等）。我们的试纸检测/设备的典型设计包括以下内容：从临床样本（如鼻拭子）中提取核酸的简单机械筒；疾病基因靶序列的快速等温扩增用简单比色法标记扩增的疾病靶序列（如染色微球）；并将标记的疾病基因序列捕获到基于三明治杂交的横向流动试纸上，以便可视化。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，以消除核酸提取的离心（或真空）要求，(2)一种新型等温核酸流感测定法，以消除这种热循环，(3)一种手持式横向流色谱试纸装置，允许对扩增产物进行多路检测/可视化。(4)一种能够同时检测/区分甲型流感、H5N1禽流感和其他几种流感样病毒SARS、RSV、副流感的综合现场流感检测装置，在一个简单的手持试纸装置中。一旦开发，我们将通过与标准RT-PCR和培养试验进行并排比较来验证临床样品的试纸试验。这种DMA量尺装置的主要用途将是在没有精密研究设施和电力的实地进行即时医疗诊断和传染病筛查（例如禽流感和SARS）。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（在一个简单的设备中可以查询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).