Rapid Detection of Agents that Cause Respiratory Disease

描述（由申请人提供）：该拟议项目的最终目标是在三年内开发一种临床诊断方法，针对15种可能被误诊为SARS病毒的呼吸道感染病原体。我们的初步目标具有由RNA或DNA构建的基因组，因此需要测试来分析两者，可能在单个试管中。MULTI-CODE平台将用于拟定的诊断。该平台操作极其简单，在不到三个小时内完成，不需要清洗，过滤昂贵的设备，并且非常适合临床实验室。 具体而言： I.该平台已在模拟实验中使用合成靶标同时检测RNA和DNA，即使靶标相差一个核苷酸。 二.该平台快速而强大。在不到三个小时内检测低拷贝数，并具有最低的“动手”要求。 三.以前，基于PCR的单重和低数量多重检测能够诊断咽拭子中的这些靶标，如本申请中所建议的。 四. EraGen在诊断平台开发（化学、分子生物学、制造）和市场实施方面的集体专业知识使其具有独特的资格来探索这项高度创新技术的能力。 第一阶段的目标是： 1.为每个靶标设计多个MULTI-CODE PCR引物和ERA-CODE引物延伸剂。 2.为至少两个内部阳性对照RNA和DNA靶点设计多个MULTI-CODE PCR引物和ERA-CODE引物延伸剂。 3.合成目标1中的所有引物和靶标。 4.确定引物和延伸剂的正确混合物，其中包括DNA和RNA内部阳性对照靶标之一，允许痰液和粪便样本的最高可能多重检测，继续允许1-100拷贝灵敏度。 5.使用多种样品制备系统，验证样品制备方法与多重测定的结合，使用加标多种组合的合成靶标的盲态对照样品。

DESCRIPTION (provided by applicant): The ultimate goal of this proposed project is to develop a clinical diagnostic in three years that targets 15 agents that cause infections in the respiratory tract that may be misdiagnosed for the SARS virus. Our preliminary targets have genomes constructed from RNA or DNA and therefore the test will be required to analyze both, possibly in a single tube. The MULTI-CODE platform will be used to for the diagnostic proposed. This platform is extremely simple to perform, is complete in less than three hours, does not require washing, filtration of expensive equipment and is uniquely suited for the clinical lab. Specifically: i. The platform has been shown in a mock experiment using synthetic targets to simultaneously detect RNA and DNA even when the targets differ by a single nucleotide. ii. The platform is fast and robust. Detecting low copy number in less than three hours with minimal "hands-on" requirements. iii. Previously, PCR based tests in both singleplex and low number multiplexes have been able to diagnose these targets in throat swabs as suggested in this application. iv. EraGen's collective expertise in diagnostic platform development (Chemistry, molecular biology, manufacturing) and market implementation makes it uniquely qualified to explore capabilities of this highly innovative technology. The Aims of Phase I are to: 1. Design multiple MULTI-CODE PCR primers and ERA-CODE primer extenders for each target. 2. Design multiple MULTI-CODE PCR primers and ERA-CODE primer extenders for at least two internal positive control RNA and DNA targets. 3. Synthesize all primers and targets from Aim 1. 4. Determine the correct mixture of primers and extenders that include one of the DNA and RNA internal positive control targets that allow for the highest multiplexing possible for both sputum and stool samples that continues to allow 1-100 copy sensitivity. 5. Using a number of sample preparation systems, validate sample preparation methods in conjunction with multiplexed assays using blinded controlled samples spiked with synthetic targets in multiple combinations.