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). Public Health Relevance Statement NARRATIVE The goal is to develop a rapid (<2h) and simple test for detection and high-resolution genetic fingerprinting of COVID-19 virus. The test is expected to resolve dozens of the COVID-19 variants that are most relevant from clinical and/or epidemiological perspectives. The test will be based on a lateral flow dipstick creating a binary barcode unique to each viral type of interest.
Project Terms: Bar Codes ; barcode ; Biotin ; Vitamin H ; coenzyme R ; Complementary DNA ; cDNA ; Chromatography ; Communities ; Containment ; Disease Outbreaks ; Outbreaks ; District of Columbia ; D.C. Washington ; DC Washington ; DNA ; Deoxyribonucleic Acid ; Epidemiology ; epidemiologic ; epidemiological ; Fingerprint ; Foundations ; Future ; Genetic Markers ; genetic biomarker ; Genome ; Goals ; Gold ; Hand ; Haplotypes ; Infection ; Laboratories ; Lead ; Pb element ; heavy metal Pb ; heavy metal lead ; Mutation ; Genetic Alteration ; Genetic Change ; Genetic defect ; genome mutation ; Nucleotides ; Oligonucleotides ; Oligo ; oligos ; Patients ; Proteins ; Ramp ; Reagent ; Reverse Transcription ; RNA ; Non-Polyadenylated RNA ; RNA Gene Products ; Ribonucleic Acid ; viral RNA ; virus RNA ; Public Health Schools ; Sensitivity and Specificity ; Technology ; Testing ; Time ; Genetic Transcription ; Gene Transcription ; RNA Expression ; Transcription ; Triplet Multiple Birth ; Triplets ; Universities ; Virus ; Washington ; Waxes ; Streptavidin ; Strepavidin ; Measures ; Healthcare ; health care ; Tube ; base ; Label ; sample collection ; specimen collection ; Lateral ; Peripheral ; Site ; 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Transmission ; Preparation ; point of care ; pandemic disease ; pandemic ; Metadata ; meta data ; genetic variant ; Gene variant ; allele variant ; allelic variant ; genomic variant ; viral detection ; virus detection ; design ; designing ; novel strategies ; new approaches ; novel approaches ; novel strategy ; Outcome ; Population ; clinically relevant ; clinical relevance ; commercialization ; phase 1 study ; Phase I Study ; phase 2 study ; phase II study ; personalized medicine ; personalization of treatment ; personalized therapy ; personalized treatment ; whole genome ; entire genome ; full genome ; novel coronavirus ; CoV emergence ; corona virus emergence ; coronavirus emergence ; emergent CoV ; emergent corona virus ; emergent coronavirus ; emerging CoV ; emerging corona virus ; emerging coronavirus ; nCoV ; new CoV ; new corona virus ; new coronavirus ; novel CoV ; novel corona virus ; 2019-nCoV ; 2019 novel corona virus ; 2019 novel coronavirus ; COVID-19 virus ; COVID19 virus ; CoV-2 ; CoV2 ; SARS corona virus 2 ; SARS-CoV-2 ; SARS-CoV2 ; SARS-associated corona virus 2 ; SARS-associated coronavirus 2 ; SARS-coronavirus-2 ; SARS-related corona virus 2 ; SARS-related coronavirus 2 ; SARSCoV2 ; Severe Acute Respiratory Distress Syndrome CoV 2 ; Severe Acute Respiratory Distress Syndrome Corona Virus 2 ; Severe Acute Respiratory Distress Syndrome Coronavirus 2 ; Severe Acute Respiratory Syndrome CoV 2 ; Severe Acute Respiratory Syndrome-associated coronavirus 2 ; Severe Acute Respiratory Syndrome-related coronavirus 2 ; Severe acute respiratory syndrome associated corona virus 2 ; Severe acute respiratory syndrome corona virus 2 ; Severe acute respiratory syndrome coronavirus 2 ; Severe acute respiratory syndrome related corona virus 2 ; Wuhan coronavirus ; coronavirus disease 2019 virus ; hCoV19 ; nCoV2 ; in silico ; SARS-CoV-2 positive ; COVID-19 positive ; COVID-19 positivity ; COVID19 positive ; COVID19 positivity ; SARS-CoV-2 positivity ; Severe acute respiratory syndrome coronavirus 2 positive ; Severe acute respiratory syndrome coronavirus 2 positivity ; coronavirus disease 2019 positive ; coronavirus disease 2019 positivity ; detection test ; detection tests ; detection limit ; SARS-CoV-2 variant ; 2019-nCoV variant ; 2019-nCoV variant forms ; 2019-nCoV variant strains ; COVID-19 variant ; COVID-19 variant forms ; COVID-19 variant strains ; SARS-CoV-2 variant forms ; SARS-CoV-2 variant strains ; coronavirus disease 2019 variant ; coronavirus disease 2019 variant forms ; coronavirus disease 2019 variant strains ; severe acute respiratory syndrome coronavirus 2 variant ; severe acute respiratory syndrome coronavirus 2 variant forms ; severe acute respiratory syndrome coronavirus 2 variant strains ; SARS-CoV-2 genome ; COVID-19 genome ; COVID-19 virus genome ; COVID19 genome ; COVID19 virus genome ; SARS-CoV2 genome ; coronavirus disease 2019 genome ; coronavirus disease 2019 virus genome ; severe acute respiratory syndrome coronavirus 2 genome ;
