Rapid point-of-care diagnostic for bioterrorism "A" agents and Pandemic influenza

生物恐怖主义“A”制剂和大流行性流感的快速护理点诊断

• 批准号: 7134823
• 负责人: Kelly J. Henrickson
• 金额: $ 160.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7134823
• 关键词: Poxviridae disease; bioterrorism /chemical warfare; clinical research; disease outbreaks; human; influenza

DESCRIPTION (provided by applicant): The terrorist attacks around the world necessitate society's continued investment in a strong defense against these unpredictable and deadly events. Infectious agents identified to pose the greatest potential threat (Category "A" agents) include Variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), and a group of RNA viruses that cause hemorrhagic fevers (VHFs). Another agent of grave concern is influenza (Flu) (Category "C"). Flu A and B viruses kill hundreds of thousands each year world wide and cost society tens to hundreds of billion dollars in morbidity and societal disruption. Additional concern exists over bird-to-human spread of Flu and the potential adaptation for human-to-human spread. Terrorist could take advantage of Flu's antigenic flexibility and engineer "shifted" more virulent strains capable of causing worldwide pandemics. Current diagnostic assays are directed to the common human isolates of Flu A (H1N1 and H3N2) and Flu B, but no assay is available to detect all of the avian antigenic varieties of Flu A (16 HA types, 9 N types). Our laboratory has pioneered a flexible, rapid, sensitive, and specific method of simultaneously detecting multiple pathogens. Our multiplex PCR-EHA tests (Hexaplex, Pneumoplex, Adenoplex, SARS/Coronaplex, etc.) are used widely around the world. We have recently developed two BioTplex assays that detect many (15) category "A" agents. However, new methods of amplified DNA detection (electronic microarrays) and nucleic acid purification now allow for the development of a single "point-of-care" device that may enhance the speed, flexibility, throughput, and cost effectiveness of multiplex assays. The Specific Aims of this application are: 1) Chemistry, we will select, optimize, and integrate sample preparation chemistry, multiplex PCR amplification systems, and electronic microarray methods to detect all antigenic variants of Flu A/B (pandemic Flu assay) and the majority of category "A" bioterrorism agents (BioT assay) in an open platform mode (separate equipment for each function); 2) Feasibility, to determine performance characteristics of the 3 modules in small pre-clinical studies using previously collected clinical samples; 3) Automation, miniaturize, integrate, and simplify the chemistry and mechanisms of SA#1 to create 3 modules and then a fully integrated single device (prototype); 4) Process development, ensure manufacturability at a reasonable cost (e.g., critical reagents, specifications, test methods, suppliers, stability, and formulation into components); 5) Pre-Clinical study, test the clinical sensitivity and specificity of the newly developed assays, compared to non-molecular methods and our "gold standard" not integrated molecular assays (PCR-EHA) on 1200 previously collected samples. These new assays, in an integrated single device, may allow cost effective, point-of-care diagnosis within 1-2 hrs in an outpatient setting. These goals are responsive to a recent NIAID announcement for High-Priority influenza (NOT-AI-05-013) and bio-threat (RFA AI-05-019) research.

描述（由申请人提供）：世界各地的恐怖袭击要求社会继续投资，以强有力地防御这些不可预测的致命事件。被确定构成最大潜在威胁的传染性病原体（“A”类病原体）包括大天花（天花）、炭疽杆菌（炭疽）、鼠疫耶尔森氏菌（鼠疫）、肉毒梭菌毒素（肉毒中毒）、土拉弗朗西斯菌（兔热病）和一组引起出血热 (VHF) 的 RNA 病毒。另一种值得严重关注的病原体是流感（流感）（“C”类）。甲型和乙型流感病毒每年在全世界造成数十万人死亡，并给社会造成数百至数千亿美元的发病率和社会破坏。人们还担心流感在鸟类之间的传播以及人际传播的潜在适应。恐怖分子可以利用流感的抗原灵活性，“改造”出能够引起全球大流行的毒性更强的毒株。 目前的诊断检测针对的是常见的人类分离的甲型流感（H1N1 和 H3N2）和乙型流感，但没有检测方法可以检测甲型流感的所有禽类抗原品种（16 种 HA 类型，9 种 N 类型）。我们实验室首创了一种灵活、快速、灵敏、特异的同时检测多种病原体的方法。我们的多重 PCR-EHA 检测（Hexaplex、Pneumoplex、Adenoplex、SARS/Coronaplex 等）在世界各地广泛使用。我们最近开发了两种 BioTplex 检测方法，可以检测许多 (15) 种“A”类药物。然而，扩增 DNA 检测（电子微阵列）和核酸纯化的新方法现在允许开发单个“现场护理”设备，该设备可以提高多重检测的速度、灵活性、通量和成本效益。此应用的具体目标是： 1) 化学，我们将选择、优化和集成样品制备化学、多重 PCR 扩增系统和电子微阵列方法，以检测 Flu A/B（大流行性流感检测）和流感病毒的所有抗原变异。大多数“A”类生物恐怖主义制剂（BioT 检测）采用开放平台模式（每个功能都有单独的设备）； 2) 可行性，使用先前收集的临床样本确定小型临床前研究中 3 个模块的性能特征； 3) 自动化、小型化、集成和简化 SA#1 的化学和机制，以创建 3 个模块，然后创建一个完全集成的单个设备（原型）； 4) 工艺开发，确保以合理的成本实现可制造性（例如关键试剂、规格、测试方法、供应商、稳定性和成分配方）； 5) 临床前研究，测试新开发的检测方法的临床敏感性和特异性，与非分子方法和我们的“金标准”非集成分子检测方法 (PCR-EHA) 相比，对 1200 个先前收集的样本进行比较。这些新的检测方法采用集成的单一设备，可以在门诊环境中 1-2 小时内实现具有成本效益的即时诊断。这些目标是对最近 NIAID 宣布的高优先级流感 (NOT-AI-05-013) 和生物威胁 (RFA AI-05-019) 研究的响应。