Rapid COVID-19 Mutation Discrimination Test for Global SARS-CoV-2 Variant Surveillance

项目摘要/摘要 公共卫生问题。新冠肺炎变异的跟踪和流行受到了极大的阻碍，因为缺乏快速、高效的 变异体检测的吞吐量方法。新冠肺炎的遗传变异是当前和持续关注的问题，原因是 更大的传播性、发病率和对疫苗提供的免疫的潜在抵抗力。成功 监测可能需要全面覆盖：100%接受测试的人(不是稀疏或地区的推断- 具体数据)。Jan Biotech提出的检测方法可以快速检测已知变种和新变种(通过检测 通过阴性结果和指示需要测序的未知序列)和探针容易 适应于检测新出现的关注和兴趣的变种。该检测将允许远程和低资源 地区医院和医疗中心快速全面评估其社区的SARS-CoV-2变异指数 实时、以证据为基础的健康要求。这既是一种紧迫的需求，也很可能是一种新的长期需求 变种出现了。 当前解决方案的问题以及产品如何满足未满足的需求。RT-PCR新冠肺炎检测仅提供 阳性或阴性结果，不识别遗传变异。新冠肺炎的快速抗体检测也不起作用 揭示变种。新冠肺炎基因组的DNA测序是具有挑战性的。基因组大约有3万个 核苷酸的长度和基因组不同区域的突变组合是有功能的 识别新冠肺炎变体的特征。用于下一代测序的高通量RNAseq方法 (NGS)需要RNA提纯、RT-PCR RNAseq文库准备和耗时的测序以及 基因组组装。用于鉴定变异的任何格式的新冠肺炎测序尚未得到CLIA-或 FDA批准的。针对不同数据挖掘的RT-qPCR分析依赖于改变的CT曲线，这些曲线是非特异性的和 可能是由化验过程中的变化引起的。提出了新冠肺炎变异的快速检测和识别方法 在多孔板中进行的测试是特定于变种的和高通量的。 方法摘要。我们将创建RNAamp寡核苷酸模板光还原探针组，专门针对 目前最普遍和具有临床意义的新冠肺炎核糖核酸变体。我们将多路传输新冠肺炎的变体 识别RNAAmp试验，对每个靶标使用不同的前荧光团，并评估灵敏度和 使用添加了多个新冠肺炎变异RNA的阴性人类唾液样本进行多重结果的可靠性。 人体样本将被用来评估多重新冠肺炎变体RNAamp测试的商业潜力。 新冠肺炎阴性样品将作为阴性对照，同样的阴性样品将添加Covid- 19个变异对照RNA将作为每个变异测试的阳性对照，以实现统计相关性 &gt；0.9，以比较分析作为成功的衡量标准。 合作者和独特资源。Jan Biotech，Inc.，在分子诊断开发方面拥有专业知识，将 从罗切斯特大学医学中心获取人类新冠肺炎阳性和阴性检测样本， 如果需要，还可以从Precision for Medicine和BocaBioltics获得。 具体目标 特异性目标1：建立新冠肺炎多重变异鉴别RNAAmp检测方法 目标1.1：开发和测试RNAAmp探针组，以区分受关注的新冠肺炎变体。 目的1.2：新冠肺炎变异区分度RNAAmp试验的多重及检验。 具体目标2：评价新冠肺炎人体样本的RNAAmp变异体识别试验 目的2.1：测试人体样本以评估新冠肺炎多重变异体RNAAmp的商业潜力。 目标2.2：统计确定每个新冠肺炎变体Will的检测下限和特异性 评估快速新冠肺炎变异鉴别检验的实用性，包括其在混合样本中的应用。 该项目的最终结果将是一个多用途的新冠肺炎变异辨别测试和计算软件 为第二阶段临床前和临床评估提供概念验证，从而实现CLIA或510(K) 批准、临床试验和商业化。

Project Summary/Abstract Public Health Problem. Covid-19 variant tracking and prevalence is greatly hindered by the lack of quick, high- throughput methods for variant detection. Covid-19 genetic variants are a current and ongoing concern, due to greater transmissibility, morbidity and potential resistance to immunity provided by vaccines. Successful surveillance will likely require full coverage: 100% of people tested (not an extrapolation of sparse or region- specific data). Jan Biotech’s proposed assay quickly detects both known variants and new variants (by detecting unknown sequences through negative results and indicating the need for sequencing) and the probes are easily adapted to detect newly emerging variants of concern and interest. The assay will allow remote and low resource area hospitals and medical centers to quickly and fully assess their community’s SARS-CoV-2 variant index for real-time, evidence-based health mandates. This is both an urgent and very likely a long term need as new variants emerge. Issues with Current Solutions & How Product Meets Unmet Needs. RT-PCR Covid-19 tests provide only a positive or negative result and do not identify genetic variants. Rapid antibody tests for Covid-19 also do not reveal variants. DNA Sequencing of the Covid-19 genome is challenging. The genome is almost 30,000 nucleotides in length and combinations of mutations in different areas of the genome are functional and identifying features of Covid-19 variants. High-throughput RNAseq methods for next-generation sequencing (NGS) require RNA purification, RT-PCR RNAseq library preparation and time-consumptive sequencing and genome assembly. Covid-19 sequencing in any format for identification of variants has not yet been CLIA- or FDA-approved. RT-qPCR assays mined for variant data rely on altered Ct curves, which are nonspecific and can be caused by variations in the assay run. The proposed rapid Covid-19 variant detection and discrimination test, performed in a multiwell plate, is variant-specific and high-throughput. Summary of Approach. We will create RNAamp oligonucleotide-templated photoreduction probe sets specific to the current most prevalent and clinically-significant Covid-19 RNA variants. We will multiplex the Covid-19 variant discrimination RNAamp tests, using different profluorophores for each target and evaluate sensitivity and reliability of multiplex results using negative human saliva samples spiked with multiple Covid-19 variant RNAs. Human samples will be used to assess commercial potential of the multiplexed Covid-19 variant RNAamp test. Covid-19 negative samples will serve as negative controls and the same negative samples spiked with Covid- 19 variant control RNAs will serve as positive controls for each variant test to achieve a statistical correlation of >0.9 with comparison assays as the metric of success. Collaborators and Unique Resources. Jan Biotech, Inc., with expertise in molecular diagnostic development, will obtain human Covid-19 positive and negative test samples from the University of Rochester Medical Center, and, as needed, from Precision for Medicine and BocaBiolistics. Specific Aims Specific Aim 1: Develop multiplexed variant discrimination RNAamp test for Covid-19 strain detection Objective 1.1: Develop and test RNAamp probe sets to differentiate Covid-19 variants of concern. Objective 1.2: Multiplex and test the Covid-19 variant discrimination RNAamp tests. Specific Aim 2: Evaluate variant discrimination RNAamp test on Covid-19 human samples Objective 2.1: Test human samples to assess commercial potential of multiplexed Covid-19 variant RNAamp. Objective 2.2: Statistical determination of assay limit of detection and specificity for each Covid-19 variant will evaluate the utility of the rapid Covid-19 variant discrimination test, including its application to pooled samples. The end result of the project will be a multiplexed Covid-19 variant discrimination test and computational software providing proof-of-concept for Phase II preclinical and clinical evaluation leading towards CLIA or 510(k) approval, clinical trials and commercialization.