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）：对流感菌株的勤奋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兴病毒。当前的监视依赖于慢慢的病毒培养，抗原识别或旨在检测已知循环亚型的分子测定法，尽管某些测定可能将新型病毒视为“未型”。 非型病毒可以是单核苷酸变体，也可以是季节性亚型的其他变体，也可以是具有大流行能力的新型替代品。当前方法缺乏轻松，准确地识别和单独跟踪这些变体的能力。该提案的目的是开发可进行现场的监视系统，该系统提供了对当前和新兴流感菌株的快速，准确的检测和序列识别，这些菌株可能会对人群造成严重的风险。该系统整合了西雅图儿童研究所（SCRI）的共识 - 基于基于PCR的杂种寡核苷酸底漆（Codehop）的PCR和一种荧光分子信标检测方法，涉及微观学的高级护理（POC）Microfluidics Sirneritics Semplostic segriptial for Pannat for pannat for pannat for Intield coppab in。监测这项技术的流感感染和新出现的流感。通过与已知的TM标志的库进行比较，可以通过季节性和大流行量来确定目前的流感病毒菌株的样本到分辨率测试的墨盒。对于新型病毒，可​​以直接对包含的Pannat弹药筒中的代码法衍生的PCR产物进行测序，以识别与新TM签名相对应的序列标签。可以通过有针对性的下一代测序对具有新TM特征鉴定的临床样本中的病毒进行完全测序，从而可以对其进化和致病性质进行全面分析，从而指导可能的治疗方法和其他公共卫生方法，以实现新出现的菌株。具体目的和里程碑包括：鼻拭子提取，cDNA转化率以及在Pannat Flu-Codehop分子测定中的扩展和歧视的发展和完全整合（AIM 1）；使用标准化的合成质粒和临床样品（AIM 2）完成了PANNAT仪器和墨盒的完成，包括用于融化曲线分析的仪器修饰和分析性能测试；以及对临床样本的前瞻性测试，并证明了与谓词测试的实质性等效性（AIM 3）。 Flu-CodeHop测定法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学威胁的呼吸道和肠道病原体或其他造成生物学威胁的药物为其他分子诊断测试提供了基础。