Point-of-care Diagnosis and Surveillance of Known and Emerging Influenza Viruses

描述（由申请方提供）：对流感病毒株进行认真的全球监测是美国国家战略和世界卫生组织（WHO）大流行性流感战略的关键要素。历史表明，需要快速识别具有大流行潜力的新出现病毒。目前的监测依赖于缓慢的病毒培养，抗原识别，或分子检测，旨在检测已知的循环亚型，虽然一些检测可能会检测新的病毒作为“未分型。“未分型的病毒可以是单核苷酸变异体或季节性亚型的其他变异体，也可以是具有大流行能力的新型病毒。在当前的方法中缺乏容易且准确地识别和单独地跟踪这些变体的能力。该提案的目标是开发一个可部署在外地的监测系统，对可能对人类构成严重威胁的现有和新出现的流感毒株进行快速、准确的检测和序列识别。该系统集成了西雅图儿童研究所（SCRI）的基于共识-简并杂交寡核苷酸引物（CODEHOP）的PCR和荧光分子信标检测方法，该检测方法在Patironics的高级护理点（POC）微流体诊断平台上，称为PanNAT”。PanNAT Flu-CODEHOP综合系统将扩大和提高流感感染和新出现的大流行病的现场和近距离患者监测能力。该技术将通过基于CODEHOP的PCR扩增和“草率”分子信标（SMB）荧光熔解曲线（Tm）特征来检测和区分流感血凝素（HA）和神经氨酸酶（NA）亚型和毒株。该检测试剂盒被整合到Panatics的PanNAT一次性检测盒中，由PanNAT仪器处理，用于样本到结果的信息点检测。目前的流感病毒株，季节性和大流行，通过与已知的Tm标签库进行比较来鉴定。可以监测导致新的独特Tm特征的新型病毒的多次发生和地理传播。对于新型病毒，PanNAT检测盒中包含的CODEHOP衍生PCR产物可直接测序，以识别与新Tm特征对应的序列标签。通过靶向Next-Gen测序，可以对临床样本中具有新Tm特征的病毒进行完全测序，从而全面分析其进化和致病特性，指导针对新出现菌株的可能治疗方法和其他公共卫生措施。具体目标和里程碑包括：在PanNAT Flu-CODEHOP分子检测中开发和全面整合鼻拭子提取、cDNA转换以及扩增子检测和区分（目标1）;完成PanNAT仪器和检测盒，包括对仪器进行修改，以进行熔解曲线分析，并使用标准化合成质粒和临床样本进行分析性能测试（目标2）;和临床样本的前瞻性试验，并证明与等同试验的实质等同性（目标3）。Flu-CODEHOP检测试剂盒的成功开发将进一步验证PanNAT微流控POC诊断平台的基础技术，为广泛的呼吸道和肠道病原体或其他对人类构成生物威胁的病原体的其他分子诊断测试提供基础。

DESCRIPTION (provided by applicant): Diligent global surveillance of influenza strains is a critical element of the U.S. National Strategy and the World Health Organization's (WHO) strategy for Pandemic Influenza. History has shown the need for rapid recognition of emerging viruses with pandemic potential. Current surveillance relies on slow virus culture, antigen recognition, or molecular assays that are designed to detect known circulating subtypes, although some assays may detect novel viruses as "untyped." Untyped viruses can be either single nucleotide variants or other variants of seasonal subtypes or novel reassortants with pandemic capabilities. The ability to easily and accurately identify and individually track these variants is lacking in current approaches. The objective of the proposal is the development of a field-deployable surveillance system that provides rapid and accurate detection and sequence recognition of current and emerging influenza strains that may present a serious risk to human populations. This system integrates Seattle Childrens Research Institute's (SCRI's) Consensus- Degenerate Hybrid Oligonucleotide Primer (CODEHOP)-based PCR and a fluorescent molecular beacon detection method on Micronics' advanced point-of-care (POC) microfluidics diagnostic platform, called the PanNAT". The integrated PanNAT Flu-CODEHOP system will expand and improve capabilities for in-field and near patient monitoring of influenza infections and emerging pandemics. This technology will detect and discriminate influenza hemagglutinin (HA) and neuraminidase (NA) subtypes and strains by means of CODEHOP-based PCR amplification and "sloppy" molecular beacon (SMB) fluorescent melt curve (Tm) signatures. The assay is incorporated into Micronics' PanNAT disposable cartridge, which is processed by the PanNAT instrument for sample-to-result point of information testing. Current influenza virus strains, both seasonal and pandemic, are identified by comparison with a library of known Tm signatures. Novel viruses resulting in new unique Tm signatures can be monitored for multiple occurrences and geographical spread. For novel viruses, the CODEHOP-derived PCR product contained in the PanNAT cartridge can be directly sequenced to identify the sequence tag corresponding to the new Tm signature. Virus in clinical samples identified with new Tm signatures can be completely sequenced via targeted Next-Gen sequencing, allowing a comprehensive analysis of its evolutionary and pathogenic properties guiding possible therapeutic approaches and other public health measures for the novel emerging strain. Specific aims and milestones include: the development and full integration of nasal swab extraction, cDNA conversion, and amplicon detection and discrimination in the PanNAT Flu-CODEHOP molecular assay (Aim 1); the completion of the PanNAT instrument and cartridges including instrument modification for melt curve analysis and analytical performance testing with standardized synthetic plasmids and clinical samples (Aim 2); and prospective testing of clinical samples and demonstration of substantial equivalence to predicate tests (Aim 3). The successful development of the Flu-CODEHOP assay will further validate the foundational technology of the PanNAT microfluidics POC diagnostic platform providing the basis for additional molecular diagnostic tests for a wide range of respiratory and enteric pathogens or other agents that pose a biological threat to humans.