Fully Automated CRISPR-LAMP Platform for SARS-CoV-2 Delta and Omicron Variants
Tong Zhang, Zhao Wang, Xi Chen, Xinlian Zhang, Jinhui Zhu, Shenwei Li, Chuanyong Wu, Zhengan Tian, Guodong Sui
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 102 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 102 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.