Abstract
Integrated clustered regularly interspaced short palindromic repeat (CRISPR)-loop-mediated amplification (LAMP) technology is of great importance in CRISPR-based diagnostic systems, which urgently needs to be developed to improve diagnostic accuracy. A labor-free, contamination-free, and fully automated droplet manipulation platform for the CRISPR-LAMP technology has not been developed before. Herein, we propose a fully automated CRISPR-LAMP platform, which can precisely manipulate the CRISPR-LAMP droplet and perform combined reactions with high sensitivity and specificity. SARS-CoV-2 Spike T478K, D614G, P681R, and P681H mutations, typical point mutations of B.1.617.2 (Delta) and Omicron variants, are monitored with this platform with a detection limit of 10 2 copies/μL. Allele discrimination between the mutants and wild type is significant with the designed one/two-mismatch CRISPR RNA (crRNA) at a limit of 10 2 copies/μL. Chemically synthesized and modified crRNAs greatly increase the CRISPR-LAMP signal, which advance the wide application. Combined with the previously developed RdRp CRISPR-LAMP assay, clinical results showed that Spike T478K and P681H can discriminate the mutant type form the wild type with 70% (49.66-85.50%, 95% confidence interval) and 78% (57.27-90.62%, 95% confidence interval) sensitivity, respectively, and 100% specificity (51.68-100%, 95% confidence interval), and the RdRp target can detect SARS-CoV-2 strains with 85% sensitivity (65.39-95.14%, 95% confidence interval) and 100% specificity (51.68-100%, 95% confidence interval). We believe that this automatic digital microfluid (DMF) system can advance the integrated CRISPR-LAMP technology with higher stability, sensitivity, and practicability, also for other CRISPR-associated diagnostic platforms.
【초록키워드】 SARS-CoV-2, spike, mutations, diagnostic, Delta, B.1.617.2, omicron, variants, RNA, CRISPR, amplification, sensitivity, specificity, stability, Accuracy, Sensitivity and specificity, RdRP, D614G, automated, mutant, P681H, P681R, wild type, platform, Detection limit, Point mutation, diagnostic systems, T478K, reaction, 95% confidence interval, SARS-CoV-2 strain, limit, Combined, IMPROVE, detect, 【제목키워드】 SARS-CoV-2, Delta, omicron,