Differentiation of Common Respiratory Viruses and SARS

2002/2003年的SARS暴发证明了新发传染病的致命性和传染性 探员们。SARS的临床表现与其他呼吸道感染相似，诊断复杂， 然而，分子诊断方法将SARS与危险性较小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，一种电池- 手提式聚合酶链式反应检测仪利用荧光检测特异性扩增产物和 该软件可自动将样品称为阳性或阴性。本申请提出了四个具体的 目标涉及将军用剃须刀转变为民用探路者，初步应用于 区分常见呼吸道病毒和非典型肺炎。具体目标I涉及对 现有仪器可实现高分辨率的热熔化曲线分析。这包括对 光学系统，使得使用新型dsDNA结合染料LC Green的检测成为可能 加热器可实现精确的温度控制。特定目标2修改爱达荷州技术公司的现有手册 从人鼻咽吸出物(选择基质)中提纯RNA的DNA纯化方法 用于诊断呼吸道感染)。特指目标3高分辨率热熔融的多重分析 曲线要求单个扩增产物的熔融特性是唯一的。一套 每种RNA呼吸道病毒的特异扩增产物：RSV、A和B流感以及SARS 冠状病毒形成时，每种病毒都有分离良好、易于分化的熔化轮廓。成套的 产生这些扩增产物的PDmer在多路复用到 4级。开发了用于执行反转录/聚合酶链式反应的冻干试剂， 是平台不可分割的一部分。具体目标4.提出将探路者集成到工作站中 自动化核酸制备并无缝地准备聚合酶链式反应。几种可能的情况 考虑了细胞裂解、核酸提取和低功率形式纯化的方法。

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 pdmers 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.