Differentiation of Common Respiratory Viruses and SARS

描述（由申请人提供）：2002/2003年的SARS爆发证明了新出现的传染性病原体的致命性和传播性。SARS的临床表现与其他呼吸道感染相似，使诊断复杂化，但通过分子手段进行诊断可将SARS与危险性较低的呼吸道感染区分开来。根据与美国国防部的合同，爱达荷州技术公司开发了RAZOR，这是一种电池供电的便携式PCR仪器，利用荧光检测特异性扩增产物，并使用软件自动将样本识别为阳性或阴性。该申请提出了四个具体目标，涉及将军用RAZOR转换为民用Pathophylla，最初应用于区分普通呼吸道病毒和SARS。具体目标I涉及对现有仪器的工程变更，以实现高分辨率的热熔融曲线分析。这包括对光学系统的改变，使得使用新型dsDNA结合染料LCGreen的检测是可能的，以及对加热器的改变，以允许精细的温度控制。Specific Aim 2修改了爱达荷州技术公司现有的手动DNA纯化方案，以从人鼻导管抽吸物样本（诊断呼吸道感染的首选基质）中纯化RNA。特定目的3高分辨率热熔解曲线的多重分析要求单个扩增产物的熔解特征是唯一的。开发了一组对每种RNA呼吸道病毒（RSV、甲型和B型流感病毒以及SARS冠状病毒）具有特异性的扩增子，每种病毒均具有良好分离且易于区分的解链谱。产生这些扩增产物的引物组以多重化至4水平所需的组合进行测试。开发了用于进行逆转录/PCR反应的冻干试剂，这些试剂是平台的组成部分。具体目标4提出将Pathology整合到工作站中，自动化核酸制备并无缝准备PCR反应。几种可能的方法，细胞裂解，核酸提取和纯化的低功率格式被认为是。

DESCRIPTION (provided by applicant): The SARS outbreak of 2002/2003 demonstrated the lethality and communicability of emerging infectious agents. Clinical presentation of SARS is similar to other respiratory infections, complicating diagnosis, however diagnosis by molecular means differentiates SARS from less dangerous respiratory infections. Under contract to the US Department of Defense, Idaho Technology developed the RAZOR, a battery-operated hand-portable PCR instrument utilizing fluorescence to detect -specific amplification products and software to automatically call samples as either positive or negative. This application proposes four specific aims involving the transforming the military RAZOR to the civilian PathFinder with an initial application to differentiate common respiratory viruses from SARS. Specific Aim I involves engineering changes to the existing instrument to allow high-resolution thermal melting curve analysis. This includes changes to the optical system such that detection using the novel dsDNA binding dye LCGreen is possible and changes to the heaters to allow fine temperature control. Specific Aim 2 modifies Idaho Technology's existing manual DNA purification protocol to purify RNA from human nasopharangeal aspirate samples (the matrix of choice for diagnosis of respiratory infections). Specific Aim 3 Multiplex analysis of high-resolution thermal melting curves requires that the melting characteristic of individual amplification products be unique. A set of amplicons specific for each of the RNA respiratory viruses: RSV, influenza A and B and the SARS coronavirus are developed with each have well-separated, readily differentiated melting profiles. Sets of primers to generate these amplification products are tested in combinations necessary for multiplexing to a level of 4. Lyophilized reagents for performing the reverse transcription/PCR reactions are developed and are an integral part of platform. Specific Aim 4 proposes to integrate PathFinder into a workstation automating nucleic acid preparation and seamlessly preparing the PCR reaction. Several possible methodologies for cell lysis, nucleic acid extraction and purification in a low power format are considered.