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

描述(由申请人提供)：勤奋的全球流感毒株监测是美国国家战略和世界卫生组织(WHO)大流行流感战略的关键要素。历史表明，需要迅速识别具有大流行潜力的新出现的病毒。目前的监测依赖于缓慢的病毒培养、抗原识别或分子检测，这些检测旨在检测已知的循环亚型，尽管一些检测可能检测到新的病毒为“未分型”。未分型病毒可以是单核苷酸变种，也可以是季节性亚型的其他变种，也可以是具有大流行能力的新型重组病毒。目前的方法缺乏轻松准确地识别和单独跟踪这些变种的能力。该提案的目标是开发一个可现场部署的监测系统，对可能对人类人口构成严重风险的当前和新出现的流感病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)基于共识退化杂交寡核苷酸引物(CODEHOP)的PCR和Microronics先进的护理点(POC)微流控诊断平台PanNAT上的荧光分子信标检测方法。集成的PanNAT Flu-CODEHOP系统将扩大和改进现场和近距离监测流感感染和新出现的大流行的能力。这项技术将通过基于CODEHOP的聚合酶链式反应扩增和“草率”分子信标(SMB)荧光熔融曲线(TM)特征来检测和区分流感血凝素(HA)和神经氨酸酶(NA)亚型和菌株。该分析被整合到Micronics的PanNAT一次性墨盒中，该墨盒由PanNAT仪器处理，用于信息测试的样本到结果点。目前的流感病毒株，无论是季节性的还是大流行的，都是通过与已知TM特征文库的比较来鉴定的。可以监测导致新的独特TM特征的新病毒的多次出现和地理传播。对于新病毒，可以直接对PanNAT盒中包含的CODEHOP衍生的PCR产物进行测序，以识别与新的TM特征相对应的序列标签。具有新TM特征的临床样本中的病毒可以通过定向下一代测序完全测序，从而能够全面分析其进化和致病特性，指导可能的治疗方法和新出现的毒株的其他公共卫生措施。具体目标和里程碑包括：开发和全面整合PanNAT Flu-CODEHOP分子分析(目标1)中的鼻拭子提取、cDNA转换、扩增检测和识别(目标1)；完成PanNAT仪器和色谱盒，包括使用标准化合成质粒和临床样本进行熔化曲线分析和分析性能测试的仪器改装(目标2)；临床样本的前瞻性测试，并证明基本上等同于预测测试(目标3)。Flu-CODEHOP检测的成功开发将进一步验证PanNAT Microfluidics POC诊断平台的基础技术，为对多种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进行额外的分子诊断测试奠定基础。

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.