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.

项目摘要/摘要 公共卫生问题。由于缺乏快速，高级，COVID-19变体跟踪和流行率极大地阻碍了 用于变体检测的吞吐量方法。 COVID-19遗传变异是当前和持续的关注，由于 疫苗提供的更大的传播，发病率和对免疫力的潜在抵抗力。成功的 监视可能需要全面覆盖：100％经过测试的人（不是稀疏或地区的外推 - 具体数据）。 JAN Biotech的拟议测定法迅速检测到已知变体和新变体（通过检测 通过负面结果未知序列，表明需要进行测序），问题很容易 适合检测新兴的关注和兴趣变体。该测定将允许远程资源 地区医院和医疗中心，以快速，充分评估其社区的SARS-COV-2变体指数 实时，基于证据的健康任务。这既是紧迫的，而且很可能是新的需求 变体出现。 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 阳性或负面结果，无法识别遗传变异。 COVID-19的快速抗体测试也不 揭示变体。 Covid-19基因组的DNA测序是挑战的。基因组接近30,000 基因组不同区域中长度和突变组合的核苷酸是功能性的，并且 识别Covid-19变体的特征。用于下一代测序的高通量RNASEQ方法 （NGS）需要RNA纯化，RT-PCR RNASEQ库制备和时间消耗的测序和 基因组组装。以任何格式的covid-19测序以识别变体的尚未是clia或clia-或 FDA批准。针对变体数据开采的RT-QPCR分析依赖于更改的CT曲线，该曲线是非特异性和 可以由测定运行中的变化引起。拟议的快速Covid-19变体检测和歧视 在多绿色板上执行的测试是特定于变异的和高通量的。 方法摘要。我们将创建RNAAMP寡核苷酸 - 模拟的光塑料探针集特有 当前最普遍和临床上最重要的Covid-19 RNA变体。我们将复用Covid-19变体 歧视RNAAMP测试，对每个目标使用不同的大量浮液，并评估灵敏度和 多重人类唾液样品的可靠性结果与多个COVID-19变体RNA峰值。 人类样品将用于评估多路复用COVID-19变体RNAAMP测试的商业潜力。 COVID-19-9负样品将用作负对照，并且与Covid-相同的负样品 19变体控制RNA将作为每个变体测试的阳性对照，以实现统计相关性 > 0.9与成功度量相比。 合作者和独特的资源。 Jan Biotech，Inc。，具有分子诊断开发方面的专业知识，将 从罗切斯特大学医学中心，获得人类共同测试样本， 并且根据需要，从医学和Bocabiolics的精度来看。 具体目标 特定目标1：开发多重变体歧视RNAAMP测试，以进行covid-19菌株检测 目标1.1：开发和测试RNAAMP探针集以区分COVID-19的关注变体。 目标1.2：多路复用并测试COVID-19变体歧视RNAAMP测试。 特定目的2：评估VoRInt Incistination RNAAMP在COVID-19的人类样品上 目标2.1：测试人类样品，以评估多路复用COVID-19变体RNAamp的商业潜力。 目标2.2：统计确定每个covid-19变体的检测和特异性的测定极限将会 评估快速Covid-19变体歧视测试的效用，包括其在汇总样品中的应用。 该项目的最终结果将是多路复用的COVID-19变体歧视测试和计算软件 提供II期临床前和临床评估的概念证明，导致CLIA或510（k） 批准，临床试验和商业化。