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，表明迫切需要即时流感分型系统;特别是因为美国超过90%的医院和较小的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做出快速反应，期望具有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，以实现细菌和/或病毒的即时分子诊断。该项目利用了在第一阶段中展示的电子微阵列平台上的芯片上的、真实的时间滚环扩增DNA的可行性，我们努力实现DNA检测的小型化和与样品制备相集成的平台的开发。以下创新解决方案形成了所提出的技术的基础：（i）在电子微阵列平台上对DNA的（分支）滚环扩增的新型实时荧光检测;（ii）结合了独特的流体学的一次性盒，其能够实现磁珠DNA/RNA提取、逆转录、DNA扩增和微阵列检测;（iii）用于电场辅助加速DNA杂交的低成本、丝网印刷的基于碳电极的微阵列。第二阶段项目的具体目标将证明该技术的可行性，包括：（1）优化碳基电子微阵列上的实时滚环扩增（RCA）条件;（2）开发和优化基于血凝素（HA）和神经氨酸酶（N）分型的流感病毒基因分型方法;（3）开发用于流感分型测定的样品制备化学品，并使用基于磁珠的RNA/DNA分离、逆转录和RCA扩增将其掺入一次性盒中;和（5）使用加标和临床（存档）样本验证流感检测。 公共卫生相关性：最近H1N1猪源流感病毒的传播和流感病毒的潜在有害突变表明迫切需要开发能够对流感病毒进行分型的即时护理系统。提供准确病毒分型的分子诊断方法将确保快速和适当的反应，并能够改善对患者的管理和限制病毒的传播。用于病毒分型的标准技术大多基于PCR或实时RT-PCR方法，这些方法复杂并且需要数小时来进行分析。该项目带来了一种新的实时DNA检测方法，该方法基于电子微阵列平台上的DNA/RNA靶标的极其快速的滚环扩增。在拟议的项目中，将全面开发仪器和检测方法，并提供一种低成本、便携式的解决方案来诊断流感和/或其他传染病。

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.