Rapid Genetic Fingerprinting of SARS-Cov-2 Variants

ABSTRACT Since the pandemic spread, SARS-Cov-2 split by mutation into several dozens of closely related clonal groups (phylogenetic clades) that continue to circulate around the globe and form sub-clades from within. Rapid point- of-care/-need capturing of the populational diversification of SARS-Cov-2 is essential for real-time surveillance, fast containment measures and personalized treatment of the patients. The goal is to develop a rapid (<2h) and simple (CLIA-moderate complexity) test for detection and high-resolution genetic fingerprinting of SARS- Cov-2 virus variants (C2F test). The test is expected to resolve a hundred or more of the SARS-Cov-2-types that are most relevant from clinical and/or epidemiological perspectives. The C2F test will be based on a novel approach of Nested Multiplex Reverse Transcription PCR (NMRTP) involving two-step reaction (virus detection and, then, fingerprint determination, both in the same reaction tube) and utilizing common laboratory thermocyclers. The fingerprint will be resolved on 10 capture lines of a lateral flow dipstick creating a binary barcode unique to each SARS-Cov-2-type of interest. First, we will select variable sites across SARS-Cov-2 genomes deposited in public database. SARS-Cov-2 genomes will be subjected to cladistic analysis to determine the main phylogenetic lineages currently circulating across USA and global regions. We will identify the most informative nucleotide positions as well as sites in SARS-Cov-2 proteins that are hotspots for mutational changes and tend to be targeted in the future. Optimal sets of target markers for genetic fingerprinting will be determined. Second, we will design multiple compatible primers for interrogation of the fingerprinting markers. We will design and test compatibility in multiplex reaction-specific primers for, on the one hand, cDNA synthesis and PCR amplifications of highly-variable regions for step 1 of the C2F test and, on the other hand, PCR amplification of the variable sites within those regions for step 2. For the purpose of primer optimization, we will utilize ~350 of SARS-Cov-2-positive oronasal samples already in hands or, if needed, recombinant synthetic SARS-Cov-2 RNA. Third, we will validate the optimized primer combinations using clinical samples. The selected primer combinations will be validated on SARS-Cov-2 positive clinical samples (e.g. oro-nasal/-pharyngeal swabs) from various patients, progressively collected during the course of study period in Seattle and Washington DC, with up to 300 samples received from each collection site. In parallel, SARS-Cov-2 genetic variants in the clinical samples will be analyzed by whole genome sequencing. Finally, we will optimize the peripheral components of the C2F test to comply with the CLIA-moderate complexity test requirements and, in Phase II, create a comprehensive database of the SARS-Cov-2 variant fingerprints and associated epidemiological and, when available, clinical metadata (e.g. asymptomatic carriage, mild or severe form of symptomatic infections, etc).