Influenza Typing Using a New Real-Time DNA Amplification on a Portable Electronic

描述（由申请人提供）：最近发生的事件，如猪流感的传播和流感病毒未来潜在的有害突变，表明迫切需要一个即时流感分型系统；特别是因为美国超过90%的医院和小型临床实验室无法进行基于dna的诊断分析。为了能够对潜在的流感爆发作出快速反应，需要有一种能够同时对流感病毒进行分型和分亚型的快速、准确的诊断方法。在样品制备、DNA/RNA提取、微流体、DNA扩增方法以及仪器小型化方面需要创新的解决方案，以使细菌和/或病毒即时分子诊断成为现实。该项目利用了芯片上的可行性，在电子微阵列平台上对DNA进行实时滚动圈扩增，我们在DNA检测小型化和开发与样品制备集成的平台方面所做的努力已在第一阶段得到证实。以下创新解决方案构成了所提出技术的基础：(i)电子微阵列平台上DNA（支链）滚动圈扩增的新型实时荧光检测；（ii）包含独特流体的一次性药筒，可实现磁珠DNA/RNA提取、逆转录、DNA扩增和微阵列检测；（iii）低成本，基于丝网印刷碳电极的微阵列，用于电场辅助加速DNA杂交。第二阶段项目的具体目标将证明该技术的可行性，该技术将包括：(1)优化碳基电子微阵列的实时滚动圈放大（RCA）条件；(2)基于血凝素（HA）和神经氨酸酶(N)分型的流感病毒基因分型方法的开发与优化；(3)开发流感分型分析的样品制备化学，并使用基于磁珠的RNA/DNA分离、逆转录和RCA扩增将样品纳入一次性药筒；(4) MDx系统开发与流感检测适应；(5)使用加标和临床（存档）样本验证流感检测。

DESCRIPTION (provided by applicant): Recent events such as the spread of swine flu and potential future detrimental mutations of influenza viruses point to an urgent need for a point-of-care influenza typing system; particularly because over 90% of hospitals and smaller clinical labs in the U.S. cannot perform DNA-based diagnostic analysis. To enable a quick response to a potential influenza outbreak, it is desirable to have a fast, accurate diagnostic method capable of simultaneously typing and subtyping influenza viruses. Innovative solutions are needed in sample preparation, DNA/RNA extraction, microfluidics, DNA amplification methods as well as instrument miniaturization to make bacterial and/or viral point-of-care molecular diagnostics a reality. This project leverages a feasibility of on-chip, real time rolling circle amplification of DNA on an electronic microarray platform demonstrated in Phase I with our efforts in miniaturization of DNA detection and development of platforms for integration with the sample preparation. The following innovative solutions form the basis of the proposed technology:(i) novel real-time fluorescence detection of (branched) rolling circle amplification of DNA on the electronic microarray platform; (ii) a disposable cartridge that incorporates a unique fluidics enabling magnetic bead DNA/RNA extraction, reverse transcription, DNA amplification and microarray detection; (iii) low cost, screen printed carbon electrodes-based microarray for electric field-assisted acceleration of DNA hybridization. The Phase II project specific aims will demonstrate the feasibility of the technology that will consist of: (1) Optimization of real-time rolling circle amplification (RCA) conditions on the carbon-based electronic microarray; (2) Development and optimization of influenza virus genotyping assay based on hemagglutinin (HA) and neuraminidase (N) typing; (3) Development of sample preparation chemistry for influenza typing assay and incorporation onto a disposable cartridge using magnetic bead based separation of RNA/DNA, reverse transcription and RCA amplification; (4) MDx system development and adaptation to influenza assay; and, (5) Validation of the influenza assay using spiked and clinical (archived) samples. PUBLIC HEALTH RELEVANCE: The recent spread of the H1N1 swine-origin influenza virus and potential detrimental mutations of influenza viruses point to an urgent need to develop point-of-care systems that will be capable of typing influenza viruses. Molecular diagnostics methods providing accurate typing of a virus will ensure a quick and appropriate response and enable improved management of patients and limit transmission of viruses. Standard techniques for viral typing are mostly based on PCR or real-time RT-PCR methods that are complex and require several hours to perform the analysis. This project brings a new real-time method for detection of DNA based on an extremely rapid rolling circle amplification of DNA/RNA targets on the electronic microarray platform. In the proposed project, the instrument and the assay will be fully developed and provide a low-cost, portable solution to diagnose influenza and/or other infectious diseases.