Novel strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harboring nucleotide changes (mutations) in the spike gene have emerged and are spreading rapidly. These mutations are associated with SARS-CoV-2 transmissibility, virulence, or resistance to some neutralizing antibodies. Thus, the accurate detection of spike mutants is crucial for controlling SARS-CoV-2 transmission and identifying neutralizing antibody-resistance caused by amino acid changes in the receptor-binding domain. Here, we developed five SARS-CoV-2 spike gene primer pairs (5-SSG primer assay; 69S, 144S, 417S, 484S, and 570S) and verified their ability to detect nine key spike mutations (ΔH69/V70, T95I, G142D, ΔY144, K417T/N, L452R, E484K/Q, N501Y, and H655Y) using a Sanger sequencing-based assay. The 5-SSG primer assay showed 100% specificity and a conservative limit of detection with a median tissue culture infective dose (TCID 50 ) values of 1.4 × 10 2 TCID 50 /mL. The accuracy of the 5-SSG primer assay was confirmed by next generation sequencing. The results of these two approaches showed 100% consistency. Taken together, the ability of the 5-SSG primer assay to accurately detect key SARS-CoV-2 spike mutants is reliable. Thus, it is a useful tool for detecting SARS-CoV-2 spike gene mutants in a clinical setting, thereby helping to improve the management of patients with COVID-19.
【초록키워드】 SARS-CoV-2, coronavirus, Mutation, Neutralizing antibodies, Sequencing, mutations, specificity, SARS-CoV-2 transmission, Transmissibility, Culture, Accuracy, L452R, spike gene, spike mutation, N501Y, management, limit of detection, Neutralizing, mutant, novel, virulence, SARS-CoV-2 spike, dose, H655Y, strain, acute respiratory syndrome, tissue, primer, clinical setting, TCID, infective, antibody-resistance, approach, helping, FIVE, Spike mutant, G142D, IMPROVE, amino acid change, detect, caused, nine, median, the receptor-binding domain, primer pair, nucleotide change, patients with COVID-19, sequencing-based assay, 【제목키워드】 Mutation, development, SARS-CoV-2 spike, sequencing-based assay,