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

用于全球 SARS-CoV-2 变异监测的快速 COVID-19 突变辨别测试

• 批准号: 10483613
• 负责人: Janet L Huie
• 金额: $ 27.58万
• 依托单位: JAN BIOTECH, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10483613
• 关键词: Rapid; COVID; 19; Mutation; Discrimination

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.

项目概要/摘要 公共卫生问题。Covid-19 变异追踪和流行率因缺乏快速、高效的方法而受到极大阻碍。 Covid-19 遗传变异的通量方法是当前持续关注的问题，因为 更大的传染性、发病率和对疫苗提供的免疫力的潜在抵抗力。 监测可能需要全面覆盖：100% 的人接受测试（不是稀疏或区域的推断） Jan Biotech 提出的检测方法可以快速检测已知变异和新变异（通过检测）。 未知序列（通过阴性结果并表明需要测序）并且探针很容易 适合检测新出现的令人关注和感兴趣的变异，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、基于证据的卫生指令既是一项紧迫的任务，也很可能是一项新的长期需求。 变种出现。 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 测试仅提供一个解决方案。 阳性或阴性结果，并且不能识别 Covid-19 的遗传变异。 揭示 Covid-19 基因组的 DNA 测序具有挑战性 基因组有近 30,000 个。 核苷酸长度和基因组不同区域的突变组合具有功能性和 Covid-19 识别变异的特征用于下一代测序。 (NGS) 需要 RNA 纯化、RT-PCR RNAseq 文库制备和耗时的测序以及 用于识别变体的任何形式的 Covid-19 测序尚未得到 CLIA 或 FDA 批准的 RT-qPCR 检测变异数据依赖于改变的 Ct 曲线，这些曲线是非特异性的。 可能是由检测运行的变化引起的。建议的快速 Covid-19 变异检测和区分。 在多孔板中进行的测试是针对变体的且高通量的。 方法总结 我们将创建特定于 RNAamp 寡核苷酸模板的光还原探针组。 当前最流行且具有临床意义的 Covid-19 RNA 变体 我们将多重 Covid-19 变体。 区分 RNAamp 测试，对每个目标使用不同的前荧光团并评估灵敏度和 使用掺有多种 Covid-19 变异 RNA 的阴性人类唾液样本进行多重结果的可靠性。 人类样本将用于评估多重 Covid-19 变异 RNAamp 测试的商业潜力。 Covid-19 阴性样本将作为阴性对照，相同的阴性样本中加入了 Covid-19 19 个变异对照 RNA 将作为每个变异测试的阳性对照，以实现以下各项的统计相关性： >0.9，以比较测定作为成功的衡量标准。 拥有分子诊断开发专业知识的合作者和独特资源。 从罗彻斯特大学医学中心获取人类 Covid-19 阳性和阴性测试样本， 并根据需要来自 Precision for Medicine 和 BocaBiolistics。 具体目标 具体目标 1：开发用于 Covid-19 菌株检测的多重变异鉴别 RNAamp 测试 目标 1.1：开发和测试 RNAamp 探针组以区分所关注的 Covid-19 变体。 目标 1.2：多重并测试 Covid-19 变异辨别 RNAamp 测试。 具体目标 2：评估 Covid-19 人类样本的变异辨别 RNAamp 测试 目标 2.1：测试人类样本以评估多重 Covid-19 变体 RNAamp 的商业潜力。 目标 2.2：对每种 Covid-19 变体的检测限和特异性进行统计测定 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多重 Covid-19 变异辨别测试和计算软件 为通往 CLIA 或 510(k) 的 II 期临床前和临床评估提供概念验证 批准、临床试验和商业化。