Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations

Abstract

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 (TCID50) values of 1.4 × 102 TCID50/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.

Fig 1. Overall schematic structures of SARS-CoV-2 spike gene and derived 5-SSG primers.

Fig 2. Sequence analysis of SARS-CoV-2 S protein.

(A) ΔH69/V70, and (B) T95I from 69S; (C) G142D, and (D) ΔY144 from 144S; (E) K417T/N, and (F) L452R from 417S; (G) E484K/Q, and (H) N501Y from 484S; (I) H655Y from 570S. Sequences showing deletions or conversions are highlighted for comparison with the Wuhan-Hu-1-CoV sequence.