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

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 and 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 rt0.9 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.