Integrated, on-chip RT-MME-PCR for influenza diagnostics; Phase 1 = sample prepar

描述（由申请人提供）：流感病毒是高度传染性的，分段负义RNA病毒，属于正粘病毒科，每年造成约114,000人住院治疗和20,000人死亡。美国食品和药物管理局（FDA）批准了几种治疗流感感染的药物，但成功的治疗取决于在发病后48小时内使用这些药物。最近在东亚暴发的H5N1禽流感进一步加剧了人们对另一次大流行的担忧，并强调在监测和诊断应用中需要进行全面的毒株分型。因此，本申请的目标是基于Akonni的凝胶元件阵列技术，开发一种集成的、样本到答案的PCR阵列，用于临床标本的流感诊断。第一阶段的具体目标是制定和实施盒式流感RNA样品制备和检测的内部标准；开发用于病毒RNA分离和纯化的样品制备子电路；并评估流感样品制备效率和改良临床样品的疗效。第一阶段的成功将导致第二阶段的提案，具体目标将是在Akonni凝胶元件阵列平台上建立流感逆转录酶、多路、微阵列增强聚合酶链反应（RT-MME-PCR）扩增方案；将样品制备和RT-MME-PCR阵列功能集成到塑料测试盒中；并在广泛的流感样本中测试和评估集成原型，包括清洁和改良的鼻拭子、咽拭子、鼻腔灌洗和/或痰临床样本，为向Quidel Corporation提交510K做准备。第二阶段结束时的性能目标是在输入目标浓度为103 TCID50 mL-1的鼻拭子、咽拭子或洗鼻液的情况下，在2小时或更短时间内获得从样本到答案的结果。集成、多路RT-MME-PCR芯片在改进的临床样品中的成功演示，将为Akonni便携式监测和诊断系统的实施奠定基础，该系统可用于快速、痕量检测和表征RNA病毒、上呼吸道病原体和感染。

DESCRIPTION (provided by applicant): Influenza viruses are highly contagious, segmented negative-sense RNA viruses that belong to the family Orthomyxoviridae, causing approximately 114,000 hospitalizations and 20,000 deaths annually. Several drugs are approved by the US Food and Drug Administration (FDA) for treating influenza infections, but successful treatment depends on administering these drugs within the first 48 h of illness. The recent outbreak of avian influenza H5N1 in East Asia has further fueled concern over another pandemic and emphasizes the need for comprehensive strain sub-typing in surveillance and diagnostic applications. The objective of this Application is therefore to develop an integrated, sample-to-answer PCR array for influenza diagnostics in clinical specimens based on Akonni's gel element array technology. Specific aims in Phase 1 are to develop and implement an internal standard for on-cartridge influenza RNA sample preparation and detection; develop a sample preparation sub-circuit for viral RNA isolation and purification; and assess influenza sample preparation efficiency and efficacy in amended clinical samples. Success in Phase 1 will result in a Phase 2 proposal where the specific aims will be to establish influenza reverse transcriptase, multiplexed, microarray-enhanced polymerase chain reaction (RT-MME-PCR) amplification protocols on the Akonni gel element array platform; integrate the sample preparation and RT-MME-PCR array functions into a plastic test cartridge; and test and evaluate the integrated prototype on an extensive panel of influenza in neat and amended nasal swabs, throat swabs, nasal lavage and/or sputum clinical samples in preparation for 510K submission with Quidel Corporation. The performance goal at the end of Phase 2 is a sample-to-answer result in 2 hours or less at an input target concentration of 103 TCID50 mL-1 nasal swab, throat swab, or nasal lavage. Successful demonstration of an integrated, multiplexed RT-MME-PCR chip in amended clinical samples will lay the foundation for implementing Akonni's portable surveillance and diagnostic system for the rapid, trace detection and characterization of RNA viruses, upper respiratory pathogens and infections. Influenza viruses are highly contagious and cause approximately 114,000 hospitalizations and 20,000 deaths annually in the U.S. alone. The continued emergence of new strains (such as influenza A H5N1) in East Asia highlights the need for rapid, comprehensive surveillance and diagnostic tools. Akonni's technology and product solutions provide a straightforward path for diagnostic detection of trace RNA viruses and infectious diseases in an integrated, sample-to-answer test cartridge and field portable instrument.