Rapid Genetic Fingerprinting of SARS-Cov-2 Variants

SARS-Cov-2 变体的快速基因指纹分析

• 批准号: 10330879
• 负责人: EVGENI Veniaminovic SOKURENKO
• 金额: $ 29.92万
• 依托单位: IDGENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330879
• 关键词: 2019-nCoV; Bar Codes; Biotin; Cellular Phone; Chromatography; Clinical; Collection; Communities; Complementary DNA; Containment; Coronavirus; DNA; Databases; Deposition; Detection; Diagnostic; Disease Outbreaks; District of Columbia; Epidemiology; Fingerprint; Foundations; Future; Genetic Fingerprintings; Genetic Markers; Genetic Transcription; Genome; Genomics; Goals; Gold; Hand; Haplotypes; Healthcare; Immunoglobulin Variable Region; Infection; Institution; Label; Laboratories; Lateral; Lead; Measures; Metadata; Mutation; Nucleotides; Oligonucleotides; Outcome; Patients; Peripheral; Phase; Phylogenetic Analysis; Population; Positioning Attribute; Preparation; Proteins; Public Health Schools; RNA; RNA Processing; Ramp; Reaction; Reagent; Recombinants; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; SARS-CoV-2 genome; SARS-CoV-2 positive; SARS-CoV-2 variant; Sampling; Sensitivity and Specificity; Severities; Side; Single Nucleotide Polymorphism; Site; Small Business Technology Transfer Research; Streptavidin; Swab; Technology; Testing; Time; Triplet Multiple Birth; Tube; Universities; Validation; Variant; Viral; Virus; Washington; Waxes; base; clinically relevant; commercialization; design; detection limit; detection test; genetic variant; genome sequencing; in silico; interest; novel; novel coronavirus; novel strategies; pandemic disease; performance tests; personalized medicine; phase 1 study; phase 2 study; point of care; portability; repository; sample collection; success; transmission process; viral RNA; viral detection; whole genome

ABSTRACT Since the pandemic spread, SARS-Cov-2 split by mutation into several dozens of closely related clonal groups (phylogenetic clades) that continue to circulate around the globe and form sub-clades from within. Rapid point- of-care/-need capturing of the populational diversification of SARS-Cov-2 is essential for real-time surveillance, fast containment measures and personalized treatment of the patients. The goal is to develop a rapid (<2h) and simple (CLIA-moderate complexity) test for detection and high-resolution genetic fingerprinting of SARS- Cov-2 virus variants (C2F test). The test is expected to resolve a hundred or more of the SARS-Cov-2-types that are most relevant from clinical and/or epidemiological perspectives. The C2F test will be based on a novel approach of Nested Multiplex Reverse Transcription PCR (NMRTP) involving two-step reaction (virus detection and, then, fingerprint determination, both in the same reaction tube) and utilizing common laboratory thermocyclers. The fingerprint will be resolved on 10 capture lines of a lateral flow dipstick creating a binary barcode unique to each SARS-Cov-2-type of interest. First, we will select variable sites across SARS-Cov-2 genomes deposited in public database. SARS-Cov-2 genomes will be subjected to cladistic analysis to determine the main phylogenetic lineages currently circulating across USA and global regions. We will identify the most informative nucleotide positions as well as sites in SARS-Cov-2 proteins that are hotspots for mutational changes and tend to be targeted in the future. Optimal sets of target markers for genetic fingerprinting will be determined. Second, we will design multiple compatible primers for interrogation of the fingerprinting markers. We will design and test compatibility in multiplex reaction-specific primers for, on the one hand, cDNA synthesis and PCR amplifications of highly-variable regions for step 1 of the C2F test and, on the other hand, PCR amplification of the variable sites within those regions for step 2. For the purpose of primer optimization, we will utilize ~350 of SARS-Cov-2-positive oronasal samples already in hands or, if needed, recombinant synthetic SARS-Cov-2 RNA. Third, we will validate the optimized primer combinations using clinical samples. The selected primer combinations will be validated on SARS-Cov-2 positive clinical samples (e.g. oro-nasal/-pharyngeal swabs) from various patients, progressively collected during the course of study period in Seattle and Washington DC, with up to 300 samples received from each collection site. In parallel, SARS-Cov-2 genetic variants in the clinical samples will be analyzed by whole genome sequencing. Finally, we will optimize the peripheral components of the C2F test to comply with the CLIA-moderate complexity test requirements and, in Phase II, create a comprehensive database of the SARS-Cov-2 variant fingerprints and associated epidemiological and, when available, clinical metadata (e.g. asymptomatic carriage, mild or severe form of symptomatic infections, etc).

抽象的 由于大流行扩散，SARS-COV-2通过突变分为几十个密切相关的克隆组 （系统发育进化枝）继续在世界各地循环并从内部循环。快速点 - SARS-COV-2的人口多样化的良好/需要捕获对于实时监视至关重要， 快速控制措施和对患者的个性化治疗。目标是发展快速（<2h） 和简单（CLIA-中度复杂性）测试用于检测和高分辨率的SARS- COV-2病毒变体（C2F测试）。预计该测试将解决一百个或更多的SARS-COV-2型 从临床和/或流行病学的角度来看，这是最相关的。 C2F测试将基于小说 涉及两步反应的嵌套多重逆转录PCR（NMRTP）的方法（病毒检测 然后，在同一反应管中的指纹测定以及使用普通实验室 热循环器。指纹将在侧向流动量的10条捕获线上解决，形成二进制 每个SARS-COV-2型感兴趣的条形码独特。首先，我们将在SARS-COV-2上选择变量站点 存放在公共数据库中的基因组。 SARS-COV-2基因组将经过cladistic分析 确定目前在美国和全球区域循环的主要系统发育谱系。我们将确定 最有用的核苷酸位置以及SARS-COV-2蛋白中的位点，这些位置是热点 突变变化并倾向于将来针对。遗传的最佳目标标记集 指纹将被确定。其次，我们将设计多个兼容引物以询问 指纹标记。我们将在多重反应特异性引物中设计和测试兼容性 一只手是C2F测试步骤1的高度变化区域的cDNA合成和PCR扩增 另一方面，第2步中这些区域内变量位点的PCR扩增。 引物优化，我们将利用〜350个SARS-COV-2阳性的Oronasal样品，或者 需要重组合成SARS-COV-2 RNA。第三，我们将验证优化的引物组合 使用临床样品。选定的底漆组合将在SARS-COV-2阳性临床上进行验证 来自各种患者的样品（例如Oro-Nasal/咽签），在此过程中逐渐收集 西雅图和华盛顿特区的学习期，每个收集地点最多收到了300个样本。在 临床样品中的平行，SARS-COV-2遗传变异将通过整个基因组测序分析。 最后，我们将优化C2F测试的外围组件 复杂性测试要求，在第二阶段中，创建了SARS-COV-2变体的全面数据库 指纹和相关的流行病学以及临床元数据（例如无症状 携带，轻度或严重的有症状感染的形式等）。