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染性。SARS的临床表现与其他呼吸道感染相似，使诊断复杂化，但通过分子手段诊断SARS可与危险性较低的呼吸道感染区分开来。根据与美国国防部签订的合同，爱达荷技术公司开发了RAZOR，这是一种电池操作的手持式便携式PCR仪器，利用荧光检测特定扩增产物和软件自动调用样品为阳性或阴性。该应用程序提出了四个具体目标，包括将军用RAZOR转变为民用PathFinder，最初的应用程序是区分普通呼吸道病毒和SARS。具体目标1涉及对现有仪器进行工程更改，以允许高分辨率热熔化曲线分析。这包括对光学系统的改变，使使用新型dsDNA结合染料LCGreen进行检测成为可能，并对加热器进行改变，以实现精细的温度控制。Specific Aim 2修改了爱达荷技术公司现有的手工DNA纯化方案，以纯化人鼻咽抽吸样本（诊断呼吸道感染的首选基质）中的RNA。高分辨率热熔化曲线的多重分析要求单个扩增产物的熔化特性是唯一的。每一种呼吸道RNA病毒（RSV、甲型流感和乙型流感以及SARS冠状病毒）都形成了一套特异性扩增子，每种扩增子都具有分离良好、易于区分的融化谱。产生这些扩增产物的引物组在多路复用至4级所需的组合中进行测试。冻干试剂执行逆转录/PCR反应被开发，是平台的一个组成部分。Specific Aim 4提出将PathFinder集成到自动化核酸制备的工作站中，无缝地准备PCR反应。几种可能的方法，细胞裂解，核酸提取和纯化在低功率格式考虑。

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.