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: 7489862
• 负责人: Kelly J. Henrickson
• 金额: $ 156.11万
• 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/7489862
• 关键词: Adenoviruses; Affect; Ambulatory Care Facilities; Animals; Anthrax disease; Antigenic Variation; Automation; Avian Influenza; Bacillus anthracis; Bacterial Pneumonia; Bathing; Benchmarking; Biological; Biological Assay; Bioterrorism; Birds; Botulinum Toxins; Botulism; Capital; Categories; Cercopithecine Herpesvirus 1; Characteristics; Chemistry; Chickenpox; Clinical; Clinical Sensitivity; Clostridium; Cyclic GMP; DNA; Detection; Development; Devices; Diagnosis; Diagnostic; Disruption; Dose; Drug Formulations; Electronics; Engineering; Ensure; Epidemic; Equipment; Event; Francisella; Francisella tularensis; Funding; Genetic; Genome; Goals; Gold; Grant; Heating; Human; Human Resources; Individual; Infectious Agent; Influenza; Investigation; Investments; Knowledge; Laboratories; Learning; Legal patent; Life; Link; Liquid substance; Mechanics; Methods; Molecular; Molecular Diagnosis; Morbidity - disease rate; National Institute of Allergy and Infectious Disease; Numbers; Outpatients; Parasites; Patients; Performance; Plague; Pliability; Polymerase Chain Reaction; Population; Preparation; Process; RNA Viruses; Reaction; Reagent; Research; Research Personnel; Sampling; Sensitivity and Specificity; Sepsis; Serotyping; Serum; Severe Acute Respiratory Syndrome; Skin; Smallpox; Societies; Specimen; Speed; Sputum; Standards of Weights and Measures; Statistically Significant; Structure; Swab; System; Taxon; Techniques; Testing; Time; Tularemia; United States Food and Drug Administration; United States National Institutes of Health; Variant; Viral; Viral Hemorrhagic Fevers; Virulent; Virus; Water; Work; Yersinia pestis; biothreat; commercialization; cost; cost effective; cost effectiveness; experience; flu; human coronavirus; influenza epidemic; instrument; killings; miniaturize; multiplex detection; nucleic acid purification; pandemic disease; pandemic influenza; pathogen; point of care; preclinical study; programs; prototype; research study; respiratory virus

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 PCRamplification 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.

世界各地恐怖袭击事件频发，社会需要持续投入强大的防御力量 对抗这些不可预测和致命的事件。传染性病原体被认为具有最大的潜力 威胁（“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 RNA 引起出血热 (VHF) 的病毒。另一种值得严重关注的病原体是流感（流感）（类别 “C”）。甲型和乙型流感病毒每年在全世界杀死数十万人，给社会造成数十至数百人的损失 数十亿美元的发病率和社会破坏。人们还担心禽流感病毒从鸟类到人类的传播 流感和人际传播的潜在适应。恐怖分子可能会利用流感 抗原灵活性和工程“转移”了能够引起世界范围内流行病的毒性更强的菌株。 目前的诊断分析针对常见的人类分离株甲型流感（H1N1 和 H3N2）和乙型流感， 但尚无检测方法可检测所有 A 型流感禽类抗原（16 种 HA 类型，9 种 N 类型）。我们的 实验室首创了一种灵活、快速、灵敏、特异的同时检测多种物质的方法 病原体。我们的多重 PCR-EHA 测试（Hexaplex、Pneumoplex、Adenoplex、SARS/Coronaplex 等）是 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) “A”类代理商。然而，扩增 DNA 检测（电子微阵列）和核酸检测的新方法 酸纯化现在允许开发单一的“现场护理”设备，可以提高速度， 多重检测的灵活性、通量和成本效益。该应用程序的具体目标是：1) 化学，我们将选择、优化和整合样品制备化学、多重PCR扩增 系统和电子微阵列方法检测流感 A/B 的所有抗原变异（大流行流感检测） 以及大多数“A”类生物恐怖主义制剂（BioT 检测）采用开放平台模式（单独的 每个功能的设备）； 2) 可行性，确定 3 个模块的性能特征 使用先前收集的临床样本进行小型临床前研究； 3）自动化、小型化、集成化、 并简化 SA#1 的化学和机制以创建 3 个模块，然后是一个完全集成的单一模块 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与非分子检测方法相比的临床敏感性和特异性 方法和我们的“金标准”未对之前收集的 1200 个样本进行整合分子测定 (PCR-EHA) 样品。这些新的检测方法，在一个集成的单一设备中，可以实现具有成本效益的即时诊断 门诊 1-2 小时内。这些目标是对最近 NIAID 宣布的高 优先流感 (NOT-AI-05-013) 和生物威胁 (RFA AI-05-019) 研究。