tetano
Editor, Senior Moderator
PLoS One
. 2021 Dec 14;16(12):e0260850.
doi: 10.1371/journal.pone.0260850. eCollection 2021.
Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations
Ho Jae Lim[SUP] 1 2 [/SUP], Min Young Park[SUP] 1 [/SUP], Hye Soo Jung[SUP] 1 [/SUP], Youngjin Kwon[SUP] 1 [/SUP], Inhee Kim[SUP] 1 [/SUP], Dong Kwan Kim[SUP] 1 [/SUP], Nae Yu[SUP] 1 [/SUP], Nackmoon Sung[SUP] 3 [/SUP], Sun-Hwa Lee[SUP] 1 [/SUP], Jung Eun Park[SUP] 2 [/SUP], Yong-Jin Yang[SUP] 1 [/SUP]
Affiliations
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.
. 2021 Dec 14;16(12):e0260850.
doi: 10.1371/journal.pone.0260850. eCollection 2021.
Development of an efficient Sanger sequencing-based assay for detecting SARS-CoV-2 spike mutations
Ho Jae Lim[SUP] 1 2 [/SUP], Min Young Park[SUP] 1 [/SUP], Hye Soo Jung[SUP] 1 [/SUP], Youngjin Kwon[SUP] 1 [/SUP], Inhee Kim[SUP] 1 [/SUP], Dong Kwan Kim[SUP] 1 [/SUP], Nae Yu[SUP] 1 [/SUP], Nackmoon Sung[SUP] 3 [/SUP], Sun-Hwa Lee[SUP] 1 [/SUP], Jung Eun Park[SUP] 2 [/SUP], Yong-Jin Yang[SUP] 1 [/SUP]
Affiliations
- PMID: 34905589
- DOI: 10.1371/journal.pone.0260850
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.