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

描述（由申请人提供）：世界各地的恐怖袭击需要社会的持续投资，对这些不可预测的和致命的事件进行强有力的防御。被确定构成最大潜在威胁的传染性病原体（“A”类病原体）包括天花病毒（天花）、炭疽杆菌（炭疽）、鼠疫耶尔森菌（鼠疫）、肉毒杆菌毒素（肉毒杆菌中毒）、土拉菌弗朗西斯菌（土拉菌病）和一组引起出血热（VHF）的RNA病毒。另一个令人严重关注的病原体是流行性感冒（流感）（C类）。甲型流感和B流感病毒每年在世界范围内杀死数十万人，并使社会在发病率和社会破坏方面损失数百亿至数千亿美元。另外，人们还担心禽流感通过鸟类传播给人类，以及禽流感可能通过人类适应性传播给人类。恐怖分子可以利用流感抗原的灵活性，设计出能够引起世界范围流行病的更致命的毒株。 目前的诊断测定针对甲型流感（H1 N1和H3 N2）和乙型流感（Flu B）的常见人类分离株，但没有测定可用于检测甲型流感的所有禽类抗原变体（16种HA型，9种N型）。我们的实验室开创了一种灵活、快速、灵敏和特异的方法，可以同时检测多种病原体。我们的多重PCR-EHA检测（Hexaplex、Pneumoplex、Adenoplex、SARS/Coronaplex等）在世界各地被广泛使用。我们最近开发了两种BioTplex检测试剂盒，可检测许多（15）类“A”试剂。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“即时”装置，其可以提高多重测定的速度、灵活性、通量和成本效益。本申请的具体目的是：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，以检测Flu A/B的所有抗原变体（大流行性流感检测）和大多数“A”类生物恐怖制剂（毕奥测定）在开放平台模式下（每个功能单独的设备）; 2）可行性，使用先前收集的临床样本在小型临床前研究中确定3个模块的性能特征; 3）自动化、简化、集成和简化SA#1的化学和机制，以创建3个模块，然后创建完全集成的单个器件（原型）;关键试剂、规格、检测方法、供应商、稳定性和组分组成）; 5）临床前研究，测试新开发的检测方法的临床灵敏度和特异性，与非分子方法和我们的“金标准”非整合分子检测方法（PCR-EHA）相比，使用1200份先前收集的样本。这些新的测定，在一个集成的单一设备，可以允许成本效益，床旁诊断在1-2小时内在门诊设置。这些目标是响应NIAID最近关于高优先级流感（NOT-AI-05-013）和生物威胁（RFA AI-05-019）研究的公告。

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.