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

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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).