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

项目总结/摘要 公共卫生问题。新型冠状病毒变种的追踪和流行受到缺乏快速、高效、 用于变体检测的通量方法。COVID-19基因变异是当前和持续的问题，由于 更高的传染性、发病率和对疫苗提供的免疫力的潜在抵抗力。成功 监测可能需要全面覆盖：100%的人接受测试（不是稀疏或区域的外推， 具体数据）。Jan Biotech提出的检测方法可以快速检测已知的变体和新的变体（通过检测 通过阴性结果检测未知序列并指示需要测序），并且探针容易 适于检测新出现的关注和感兴趣的变体。该检测试剂盒将允许远程和低资源 地区医院和医疗中心迅速全面评估其社区的SARS-CoV-2变异指数， 实时的、基于证据的健康指令。这既是一个紧迫的，也很可能是一个长期的需要，因为新的 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 Covid-19检测仅提供 阳性或阴性结果，不能识别遗传变异。新冠肺炎快速抗体检测也不 显示变量。2019冠状病毒病基因组的DNA测序具有挑战性。基因组几乎有3万个 在基因组的不同区域中的长度和突变组合的核苷酸是功能性的， 识别新冠病毒变种的特征。用于下一代测序的高通量RNAseq方法 (NGS)需要RNA纯化、RT-PCR RNAseq文库制备和耗时测序， 基因组组装用于识别变体的任何形式的Covid-19测序尚未被CLIA-或 FDA批准的用于变异数据的RT-qPCR测定依赖于改变的Ct曲线，其是非特异性的， 可能是由测定运行中的变化引起的。拟议的快速Covid-19变异检测和鉴别 在多孔板中进行的测试是变体特异性和高通量的。 方法总结。我们将创建RNAamp寡核苷酸为模板的光还原探针组， 目前最流行和临床上最重要的Covid-19 RNA变体。我们将把新型冠状病毒变种 区分RNAamp测试，使用不同的profluorophores为每个目标，并评估灵敏度， 使用加标多种Covid-19变异RNA的阴性人唾液样本的多重结果的可靠性。 人类样本将用于评估多重Covid-19变异体RNAamp检测的商业潜力。 Covid-19阴性样本将作为阴性对照，加标Covid-19的相同阴性样本将作为阴性对照。 19种变体对照RNA将用作每种变体测试的阳性对照，以实现以下统计学相关性： >0.9，比较测定作为成功的度量。 合作伙伴和独特的资源。Jan Biotech，Inc.，拥有分子诊断开发方面的专业知识， 从罗切斯特大学医学中心获得人类新冠病毒阳性和阴性检测样本， 并根据需要，从精准医学和博卡生物技术。 具体目标 具体目标1：开发用于Covid-19毒株检测的多重变异体鉴别RNAamp检测 目标1.1：开发和测试RNAamp探针组，以区分关注的Covid-19变体。 目的1.2：多重检测和测试Covid-19变异体识别RNAamp检测。 具体目标2：评估新冠病毒人类样本的变异辨别RNAamp检测 目的2.1：测试人类样本，以评估多重Covid-19变异体RNAamp的商业潜力。 目标2.2：统计确定每种Covid-19变异体的检测限和特异性， 评估快速Covid-19变异辨别测试的实用性，包括其在合并样本中的应用。 该项目的最终结果将是一个多路复用的Covid-19变异辨别测试和计算软件 为导致CLIA或510（k）的II期临床前和临床评价提供概念验证 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.