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).

摘要 自大流行传播以来，SARS-Cov-2通过突变分裂成数十个密切相关的克隆群 （系统发育分支）继续在地球仪周围循环并从内部形成亚分支。快速点- 对SARS-Cov-2人群多样化的护理/需求捕获对于实时监测是必不可少的， 快速控制措施和个性化治疗的病人。目标是开发快速（<2 h） 和简单的（CLIA-中等复杂性）检测和SARS的高分辨率基因指纹- Cov-2病毒变体（C2F测试）。该测试预计将解决100个或更多的SARS-Cov-2类型 从临床和/或流行病学的角度来看最相关。C2F测试将基于一部小说 一种包括两步反应（病毒检测）的巢式多重逆转录PCR（NMRTP）方法 然后，指纹测定，两者都在同一反应管中）和利用共同的实验室 热循环仪。指纹将在侧向流动量油尺的10条捕获线上解析，从而产生二进制 每个SARS-Cov-2类型的唯一条形码。首先，我们将在SARS-Cov-2中选择可变位点 保存在公共数据库中的基因组。SARS-Cov-2基因组将进行分支分析， 确定目前在美国和全球地区流行的主要系统发育谱系。我们将确定 SARS-Cov-2蛋白中最具信息性的核苷酸位置以及作为SARS-Cov-2蛋白的热点的位点， 突变的变化，并倾向于在未来的目标。遗传标记的最佳靶标记组 指纹将被确定。其次，我们将设计多个相容性引物用于询问 指纹标记我们将设计和测试多重反应特异性引物的相容性， 一方面，C2F测试步骤1的高可变区的cDNA合成和PCR扩增， 另一方面，对于步骤2，PCR扩增那些区域内的可变位点。为目的 引物优化，我们将利用约350个SARS-Cov-2阳性口鼻样本， 需要的，重组合成的SARS-Cov-2 RNA。第三，我们将验证优化的引物组合 使用临床样本。所选择的引物组合将在SARS-Cov-2阳性临床试验中进行验证。 来自不同患者的样本（例如口鼻/咽拭子），在治疗过程中逐步收集 研究期间在西雅图和华盛顿DC，从每个采集点收到多达300份样本。在 与此同时，将通过全基因组测序分析临床样本中的SARS-Cov-2遗传变异。 最后，我们将优化C2F测试的外围组件，以符合CLIA-中度 复杂性测试要求，并在第二阶段建立一个全面的SARS-Cov-2变异数据库 指纹和相关的流行病学，以及临床元数据（如无症状 携带、轻微或严重形式的有症状感染等）。