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

已知和新出现的流感病毒的即时诊断和监测

• 批准号: 8820237
• 负责人: JOHN Charles GERDES
• 金额: $ 108.37万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8820237
• 关键词: Amino Acid Motifs; Antigens; Archives; Biological; Biological Assay; Birds; Categories; Cessation of life; Child; Classification; Clinical; Collaborations; Complementary DNA; Computer software; Consensus; Country; Databases; Detection; Development; Diagnosis; Diagnostic; Discrimination; Elements; Epidemiology; Flu virus; Funding; Genes; Genome; Genotype; Guidelines; Health; Hemagglutinin; Human; Hybrids; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H7N9 Subtype; Influenza A virus; Influenza B Virus; Influenza Hemagglutinin; Libraries; Measures; Methods; Mexico; Microfluidics; Modification; Molecular; Molecular Diagnostic Testing; Monitor; National Institute of Allergy and Infectious Disease; Neuraminidase; Nose; Nucleotides; Oligonucleotide Primers; Patient Monitoring; Patients; Plasmids; Population; Process; Property; Public Health; RNA; Recording of previous events; Research Institute; Reverse Transcription; Risk; Rosa; Sampling; Sensitivity and Specificity; Specimen; Swab; Symptoms; System; Technology; Test Result; Testing; Therapeutic; United States National Institutes of Health; Validation; Variant; Viral; Viral Genome; Virus; Washington; World Health Organization; base; cost effective; design; diagnostic assay; enteric pathogen; flu; genome sequencing; improved; influenza outbreak; influenza virus strain; influenzavirus; instrument; melting; mortality; next generation sequencing; novel; novel virus; pandemic disease; pandemic influenza; performance tests; point of care; point-of-care diagnostics; prospective; public health intervention; research clinical testing; research study; respiratory; swine flu; virus culture; wild bird

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.

描述（由申请人提供）：勤勉的全球流感毒株监测是美国国家战略和世界卫生组织（WHO）大流行性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病毒。目前的监测依赖于慢速病毒培养、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“未分型”的新型病毒。无型病毒可以是单核苷酸变异或季节性亚型的其他变异，也可以是具有大流行能力的新型重组病毒。目前的方法缺乏简单、准确地识别和单独跟踪这些变体的能力。该提案的目标是开发一个可在实地部署的监测系统，对可能对人群构成严重风险的当前和新出现的流感毒株进行快速和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-简并杂化寡核苷酸引物（CODEHOP）的PCR和microics先进的即时（POC）微流体诊断平台（称为PanNAT）上的荧光分子信标检测方法。综合PanNAT流感- codehop系统将扩大和改进现场和近病人监测流感感染和新出现的大流行的能力。该技术将通过基于codehop的PCR扩增和“草率”分子信标（SMB）荧光熔融曲线（Tm）特征检测和区分流感血凝素（HA）和神经氨酸酶（NA）亚型和菌株。该检测并入Micronics的PanNAT一次性药筒，由PanNAT仪器进行样品到结果点信息测试。通过与已知Tm特征库的比较，确定了当前的季节性和大流行流感病毒株。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试剂盒中含有的cocodehop衍生PCR产物可以直接测序，以确定与新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靶向Next-Gen测序进行完全测序，从而允许对其进化和致病特性进行全面分析，指导可能的治疗方法和针对新出现的菌株的其他公共卫生措施。具体目标和里程碑包括：在PanNAT流感- codehop分子分析中开发和完全整合鼻拭子提取、cDNA转换和扩增子检测和区分（目标1）；完成PanNAT仪器和药盒，包括对熔体曲线分析和标准化合成质粒和临床样品分析性能测试的仪器进行修改（目标2）；临床样本的前瞻性测试和证明与谓词测试的实质等同（目标3）。流感- codehop检测的成功开发将进一步验证PanNAT微流体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提供额外的分子诊断测试基础。