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

世界各地的恐怖袭击需要社会继续投资于强大的防御 对抗这些不可预测和致命的事件。被确定为构成最大潜力的感染性病原体 威胁(“A”类制剂)包括主要天花(天花)、炭疽杆菌(炭疽)、鼠疫耶尔森菌。 (鼠疫)、肉毒梭菌毒素(肉毒杆菌中毒)、图拉氏方济氏菌(图拉拉血症)和一组RNA 引起出血热(VHFs)的病毒。另一个令人严重关切的病原体是流感(流感)(类别 “C”)。甲型和乙型流感病毒每年在全球范围内导致数十万人死亡，给社会造成数万至数百人的损失 造成数十亿美元的发病率和社会混乱。另一个令人担忧的问题是禽流感从鸟到人的传播 流感和人与人之间传播的潜在适应。恐怖分子可能会利用流感 抗原性的灵活性和工程“转移”了能够引起全球大流行的更强的毒力菌株。 目前的诊断分析针对的是人类常见的甲型流感(H1N1和H3N2)和乙型流感分离株， 但目前还没有检测到所有禽流感病毒(16个HA型，9个N型)的检测方法。我们的 实验室率先开发了一种灵活、快速、灵敏和特异的同时检测多个 病原体。我们的多重PCR-EHA检测(六联、肺泡、腺泡、SARS/冠状等)是 在世界各地广泛使用。我们最近开发了两种BioTplex检测方法，可以检测到许多(15) “A”类特工。然而，扩增DNA检测的新方法(电子微阵列)和核 酸净化现在允许开发一种可提高速度的单一的“护理点”装置， 多重分析的灵活性、吞吐量和成本效益。本申请的具体目的是：1) 化学，我们将精选、优化和整合样品前处理化学、多重PCR扩增 检测甲型流感/乙型流感所有抗原变异的系统和电子微阵列方法(大流行性流感化验) 以及大多数A类生物恐怖主义药物(Biot试剂法)在开放平台模式下(单独 每个功能的设备)；2)可行性，以确定以下3个模块的性能特征 使用以前收集的临床样本进行的小型临床前研究；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，然后是完全集成的单个 设备(原型)；4)工艺开发，确保可制造性，成本合理(例如，关键 试剂、规格、试验方法、供应商、稳定性和配方组成)；5)临床前 研究、测试新开发的检测方法与非分子检测方法的临床敏感性和特异性 方法和我们的“金标准”非整合分子分析(PCR-EHA)对1200个先前收集的数据 样本。这些新的分析，在一个集成的单一设备中，可以实现经济高效的医疗保健诊断 在门诊部的1-2小时内。这些目标是对NIAID最近宣布的High- 优先流感(非-AI-05-013)和生物威胁(RFA AI-05-019)研究。

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.