Rapid Field Appropriate Diagnostics for Select Biodefense and Emerging Pathogens

昂贵的设备、训练有素的人员以及对临床实验室环境的需求 排除了大多数正在开发中的传染病的常规核酸测试(NAT) 世界，甚至在美国许多资源有限的地区，导致了巨大的差距 在世界各地的医疗保健方面。一种快速、灵敏、低成本但简便易行的健壮检测NAT方法 在护理点(POC)使用特定试剂将有助于将分子诊断带给每个人。 本应用程序的目标是演示完全适用于现场的NAT的可行性 用于检测病毒出血热(VHF)RNA的诊断系统，用于低资源设置。 作为这一应用的基础的创新技术是一种新的热稳定性 具有天然逆转录酶(RT)活性的聚合酶称为PYROSCRIPT。PyroScrip可以 执行一种称为LOOP的聚合酶链式反应(PCR)的有前景的NAT替代方案 中介等温扩增(LAMP)。由于灯是等温的，所以不需要 专门的仪器加上它比聚合酶链式反应快得多。LAMP对抑制剂也有抵抗力 在粗制样品制备中。PYROSCRIPT聚合酶是唯一已知的耐热性 结合链置换活性的LAMP扩增和RT酶 直接从RNA扩增的活性。这种酶的技术优势是： 1)单一稳定的酶不同于需要两个或更多不稳定的酶来检测RNA的方法。 2)嗜热的火焰石可以在95℃下变性。 3)用PyroScrip对RNA进行快速等温扩增，时间不超过30分钟。 拟议研究的目标是进一步改进PyroScrip NAT试剂 可提供现场稳定性性能的配方。另一个目标是开发一种核子 酸样制备方法简单易用，适合护理点实施。 这些创新将被合并到当地相关的诊断小组中，这些小组可以 检测某一特定地区特有的病原体。 通过将Lucigens在扩增和检测方面的能力与VHF的专业知识相结合 加尔维斯顿国家实验室提供的研究和基本能力 将开发用于检测病毒RNA的系统，该系统几乎可以在任何地方实施， 全世界。

Expensive equipment, highly-trained personnel, and the need for a clinical laboratory setting precludes routine nucleic acid testing (NAT) for infectious disease in most of the developing world and even in many resource-limited parts of the United States, leading to wide disparities in health care worldwide. A fast, sensitive, low cost but facile NAT method for robust detection of specific agents at point of care (POC) would help bring molecular diagnostics to everyone. The goal of this application is to demonstrate feasibility of a complete field-appropriate NAT diagnostics system for detection of viral hemorrhagic fever (VHF) RNA for low resource settings. The innovative technology that is the basis of this application is a new thermostable polymerase with innate reverse transcriptase (RT) activity called PyroScript. PyroScript can perform a promising NAT alternative to the polymerase chain reaction (PCR) called loop mediated isothermal amplification (LAMP). Since LAMP is isothermal it does not require specialized instrumentation plus it is much faster than PCR. LAMP is also resistant to inhibitors in crude sample preparations. The PyroScript polymerase is the only known thermostable enzyme combining both strand displacement activity for LAMP-based amplification and RT activity to amplify directly from RNA. The technical advantages of this enzyme are: 1) A single stable enzyme unlike methods requiring two or more labile enzymes to detect RNA. 2) Thermophilic PyroScript can be denatured at 95¿C. 3) Rapid isothermal amplification from RNA with PyroScript in under 30 minutes. The goal of the proposed research is to further improve PyroScript NAT reagent formulations to provide field-capable stability performance. Another goal is to develop a nucleic acid sample preparation method with ease of use suitable for point of care implementation. These innovations will be combined into locally relevant diagnostic panels that can be performed to test for pathogens endemic to a specific region. By combining Lucigens capacity in amplification and detection with the expertise in VHF research and fundamental capability provided by the Galveston National Laboratory a NAT system will be developed for detection of viral RNA that can be implemented almost anywhere, worldwide.