Rapid Genetic Fingerprinting of SARS-Cov-2 Variants

摘要 自疫情蔓延以来，SARS-CoV-2通过突变分裂成数十个密切相关的克隆群 (系统发育支系)，继续在全球范围内循环，并从内部形成亚支。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， 快速遏制措施和对患者的个性化治疗。我们的目标是开发一个快速(&lt；2小时) 和简单(CLIA-中等复杂性)检测和高分辨率SARS基因指纹分析- CoV-2病毒变异株(C2F试验)。这项检测预计将解决100或更多的SARS-CoV-2类型 从临床和/或流行病学角度来看最相关的疾病。C2F测试将基于一种新的 套式多重逆转录聚合酶链式反应(NMRTP)两步法(病毒检测) 然后，指纹测定，两者都在同一反应管中)并利用普通实验室 热循环器。指纹将在创建二进制的横向流动试纸的10条捕获线上进行解析 每个感兴趣的SARS-CoV-2类型的唯一条形码。首先，我们将选择SARS-CoV-2的可变位点 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以 确定目前在美国和全球地区流行的主要系统发生谱系。我们将确定 SARS-CoV-2蛋白中信息量最大的核苷酸位置和位点是 突变的变化，并倾向于在未来的目标。遗传标记的最优靶标组合 指纹识别将被确定。其次，我们将设计多个相容的引物用于审讯 指纹标记。我们将设计和测试多重反应特定引物的兼容性，用于 一方面，C2F测试步骤1的高可变区的cDNA合成和PCR扩增，以及 另一方面，在步骤2中，对这些区域内的可变区进行PCR扩增。 引物优化，我们将利用手中已有的约350份SARS-CoV-2阳性口腔样本，如果 所需的重组合成SARS-CoV-2 RNA。第三，对优化后的引物组合进行验证 使用临床样本。所选择的引物组合将在SARS-CoV-2阳性临床上进行验证 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 在西雅图和华盛顿特区的研究期间，从每个采集点收到多达300个样本。在……里面 同时，临床样本中的SARS-CoV-2基因变异将通过全基因组测序进行分析。 最后，我们将优化C2F测试的外围组件以符合CLIA-Medium 复杂性测试要求，并在第二阶段创建SARS-CoV-2变种的全面数据库 指纹和相关的流行病学数据，以及临床元数据(如无症状 携带、轻微或严重形式的症状性感染等)。

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).