Rapid Field Appropriate Diagnostics for Select Biodefense and Emerging Pathogens

针对特定生物防御和新兴病原体的快速现场适当诊断

• 批准号: 9068657
• 负责人: Dipankar Manna
• 金额: $ 44.56万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068657
• 关键词: Address; Chemistry; Clinical; Communicable Diseases; Complementary DNA; Detection; Devices; Diagnostic; Discrimination; Enzymes; Equipment; Formulation; Goals; Healthcare; Heating; Human; Human Resources; Incubated; Infectious Agent; Laboratories; Lateral; Mediating; Methods; Nucleic Acid Amplification Tests; Nucleic Acids; Performance; Phase; Polymerase; Polymerase Chain Reaction; Preparation; Process; RNA; RNA amplification; RNA-Directed DNA Polymerase; Reaction; Reagent; Research; Resistance; Resources; Sampling; Solid; Specimen; System; Temperature; Testing; Training; Tube; United States; Viral; Viral Hemorrhagic Fevers; amplification detection; base; biodefense; cost; design; diagnostic panel; fundamental research; health disparity; improved; inhibitor/antagonist; innovation; innovative technologies; instrumentation; molecular diagnostics; nonhuman primate; pathogen; point of care; prevent; viral RNA

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.

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 在大多数发展中国家， 世界上，甚至在美国的许多资源有限的地区， in health健康care保健worldwide世界.一种快速、灵敏、低成本但简便的NAT方法，用于稳健检测 在护理点（POC）的特定试剂将有助于将分子诊断带给每个人。 此应用程序的目标是证明一个完整的现场适当的NAT的可行性 用于检测病毒性出血热（VHF）RNA的诊断系统，用于低资源环境。 作为该应用基础的创新技术是一种新型热稳定 聚合酶具有先天逆转录酶（RT）活性，称为PyroScript。PyroScript可以 执行一个有前途的NAT替代聚合酶链式反应（PCR）称为环 介导等温扩增（LAMP）。由于LAMP是等温的， 而且它比PCR快得多。LAMP也对抑制剂有抗性 在粗样品制备中。PyroScript聚合酶是唯一已知的热稳定聚合酶。 结合了用于基于LAMP的扩增和RT的链置换活性的酶 直接从RNA扩增的活性。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。 本研究的目的是进一步改进PyroScript NAT试剂 制剂以提供能够现场使用的稳定性性能。另一个目标是开发一种 酸样品制备方法，易于使用，适用于现场护理实施。 这些创新将结合到当地相关的诊断面板， 用来检测特定地区特有的病原体。 通过结合卢西根的能力，在放大和检测的专业知识，在甚高频 由加尔维斯顿国家实验室提供的研究和基础能力 将开发用于检测病毒RNA的系统， 国际吧