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

描述(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可能有害的突变，表明迫切需要一种护理点流感分型系统；特别是因为美国90%以上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暴发做出快速反应，人们希望有一种快速、准确的诊断方法，能够同时对流感病毒进行分型和分型。在样品制备、DNA/RNA提取、微流控、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/或病毒护理点分子诊断成为现实。该项目利用了在第一阶段演示的芯片上的DNA实时滚动圆扩增的可行性，我们致力于DNA检测的小型化和与样品制备集成的平台的开发。以下创新解决方案构成了拟议技术的基础：(I)新型实时荧光检测(分支)滚动圆圈DNA在电子微阵列平台上的扩增；(Ii)一次性墨盒，其结合了独特的流体，能够实现磁珠DNA/RNA提取、反转录、DNA扩增和微阵列检测；(Iii)低成本、丝网印刷碳电极微阵列，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)碳基电子微阵列实时滚环扩增(RCA)条件的优化；(2)基于血凝素(HA)和神经氨酸酶(N)配型的流感病毒基因分型方法的开发和优化；(3)用于流感分型分析的样品制备化学的开发，并通过基于磁珠的RNA/DNA分离、反转录和RCA扩增将其整合到一次性墨盒上；(4)MDX系统的开发和适应流感检测；以及，(5)使用添加的和临床(存档)样本对流感检测进行验证。 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护理点系统。分子诊断方法提供准确的病毒分型，将确保快速和适当的反应，并能够改进对患者的管理，限制病毒的传播。病毒分型的标准技术大多基于聚合酶链式反应或实时RT-PCR方法，这些方法很复杂，需要几个小时才能进行分析。本项目提出了一种新的实时检测DNA的方法，该方法基于电子微阵列平台上DNA/RNA靶标的极快速滚动循环扩增。在拟议的项目中，仪器和化验将得到充分开发，并提供一种低成本、便携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.