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

描述(申请人提供)：世界各地的恐怖袭击需要社会继续投资，以强大的防御这些不可预测的和致命的事件。被确定为构成最大潜在威胁的传染病病原体(A类病原体)包括天花(天花)、炭疽杆菌(炭疽)、鼠疫耶尔森氏菌(鼠疫)、肉毒杆菌毒素(肉毒杆菌中毒)、图拉氏菌(图拉氏菌病)和一组引起出血热的核糖核酸病毒(VHFs)。另一个令人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球范围内导致数十万人死亡，并给社会造成数百亿美元的发病率和社会混乱。对禽流感在禽类到人类之间的传播以及可能适应人与人之间的传播也存在额外的担忧。恐怖分子可以利用流感的抗原性灵活性，设计出能够引发全球大流行的毒力更强的毒株。 目前的诊断检测针对的是人类常见的甲型流感病毒(H1N1和H3N2型)和乙型流感病毒，但没有检测到甲型流感病毒的所有禽类抗原变种(16种HA型，9种N型)。本实验室开创了一种灵活、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(六联、肺泡、腺泡、SARS/冠状等)在世界各地被广泛使用。我们最近开发了两种BioTplex检测方法，可以检测许多(15)类“A”病毒。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一种单一的“护理”设备，可以提高多重分析的速度、灵活性、吞吐量和成本效益。这项应用的具体目标是：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，以开放平台模式(每个功能分别使用单独的设备)检测流感A/B(大流行流感检测)和大多数A类生物恐怖主义药物(Biot检测)的所有抗原变体；2)可行性，利用以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，然后完全集成单个设备(原型)；4)流程开发，以合理的成本确保可制造性(例如关键试剂、规格、测试方法、供应商、稳定性和配方)；5)临床前研究，测试新开发的分析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”非整合分子分析(PCR-EHA)相比，对1200个先前收集的样本进行测试。这些新的分析，在一个集成的单一设备中，可能允许在门诊环境中在1-2小时内进行经济高效的护理点诊断。这些目标是对NIAID最近宣布的高优先级流感(非-AI-05-013)和生物威胁(RFA AI-05-019)研究的回应。

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.