Rapid Field Appropriate Diagnostics for Select Biodefense and Emerging Pathogens

描述（由申请人提供）：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使得大多数发展中国家甚至美国许多资源有限的地区无法进行传染病的常规核酸检测（NAT），从而导致全球医疗保健的巨大差异。一种快速、灵敏、低成本但简便的NAT方法，用于在护理点（POC）检测特定试剂，将有助于为每个人提供分子诊断。此应用程序的目标是证明可行性 一个完整的现场适当的NAT诊断系统，用于检测病毒性出血热（VHF）RNA的低资源设置。 作为该应用基础的创新技术是一种具有先天逆转录酶（RT）活性的新型热稳定聚合酶，称为PyroScript。PyroScript可以执行一种有前途的NAT替代聚合酶链反应（PCR），称为环介导等温扩增（LAMP）。由于LAMP是等温的，它不需要专门的仪器，而且它比PCR快得多。LAMP还对粗样品制备物中的抑制剂具有抗性。PyroScript聚合酶是唯一已知的热稳定酶，其结合了用于基于LAMP的扩增的链置换活性和直接从RNA扩增的RT活性。这种酶的技术优势是： 1)一种稳定的酶，不像需要两种或更多种不稳定的酶来检测RNA的方法。 2)嗜热PyroScript可在95 º C下变性。 3)使用PyroScript在30分钟内快速等温扩增RNA。拟议研究的目标是进一步改进PyroScript NAT试剂配方，以提供现场可用的稳定性性能。另一个目标是开发一种易于使用的核酸样品制备方法，其适用于现场护理实施。这些创新将结合到当地相关的诊断小组中，可以进行特定地区流行的病原体检测。通过将Lucigen在扩增和检测方面的能力与VHF研究方面的专业知识和加尔维斯顿国家实验室提供的基本能力相结合，将开发一种NAT系统，用于检测病毒RNA，该系统几乎可以在世界任何地方实施。 公共卫生相关性：快速、灵敏、低成本但简便的核酸测试（NAT）方法用于在护理点稳健地检测特异性试剂，这将使分子诊断的能力达到低资源设置。

DESCRIPTION (provided by applicant): 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 cos 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 Lucigen's 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. PUBLIC HEALTH RELEVANCE: A fast, sensitive, low cost but facile nucleic acid test (NAT) method for robust detection of specific agents at point of care would bring the power of molecular diagnostics to low resource settings.