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 对抗这些不可预测的致命事件已确定的最有可能造成 威胁（“A”类制剂）包括天花、炭疽杆菌、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（土拉菌血症），和一组RNA 引起出血热（VHF）的病毒。另一个令人严重关注的病原体是流行性感冒（流感）（ “C”）。甲型流感和B流感病毒每年在全世界造成数十万人死亡，并使社会损失数十至数百人 数十亿美元的发病率和社会破坏。对鸟类向人类传播禽流感的担忧 流感和人与人之间传播的潜在适应。恐怖分子可以利用流感 抗原灵活性和工程“改变”了能够引起世界范围大流行的更强毒的菌株。 目前的诊断测定针对甲型流感（H1N1和H3 N2）和乙型流感（B）的常见人类分离株， 但是没有检测甲型流感病毒的所有禽类抗原种类（16种HA型，9种N型）的方法。我们 实验室开创了一种灵活、快速、灵敏和特异的方法， 病原体我们的多重PCR-EHA检测（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，可检测许多（15） A类特工然而，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可提高速度的单一“护理点”装置， 多重检测的灵活性、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 检测甲型/B流感病毒所有抗原变体的系统和电子微阵列方法（大流行性流感检测试剂盒） 和大多数“A”类生物恐怖剂（毕奥分析）以开放平台模式（单独的 2）可行性，以确定3个模块的性能特征， 使用先前收集的临床样品的小型临床前研究; 3）自动化、自动化、集成， 并简化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模块， 设备（原型）; 4）工艺开发，确保以合理的成本可制造（例如，关键 试剂、规格、试验方法、供应商、稳定性和组分配制）; 5）临床前 研究，测试新开发的检测试剂盒的临床灵敏度和特异性，与非分子检测试剂盒相比， 方法和我们的“金标准”不整合分子检测（PCR-EHA）对1200先前收集的 样品在集成的单个设备中，这些新的测定可以允许具有成本效益的即时诊断。 在门诊1-2小时内。这些目标是响应最近NIAID宣布的高- 优先流感（NOT-AI-05-013）和生物威胁（RFA AI-05-019）研究。

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.