A rapid screening assay for L452R and T478K spike mutations in SARS-CoV-2 Delta variant using high-resolution melting analysis

J Toxicol Sci. 2021;46(10):471-476. doi: 10.2131/jts.46.471.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission has been reported worldwide and novel SARS-CoV-2 variants continue to emerge. A novel SARS-CoV-2 strain, the Delta variant (B.1.617.2), is spreading worldwide. The Delta variant has reportedly high infectivity and immune evasion potency. In June 2021, the World Health Organization categorized it as a variant of concern (VOC). Therefore, it is vital to develop tests that can exclusively identify the Delta variant. Here, we developed a rapid screening assay to detect characteristic mutations observed in the Delta variant using high-resolution melting (HRM) analysis. In this assay, we determined L452R and T478K, among which T478K is an identifier of the Delta variant since L452R is seen in other strains (Kappa and Epsilon variants). Additionally, nested PCR-based HRM analysis, which involved RT-PCR (1st PCR) and HRM analysis (2nd PCR), was developed to improve the specificity and sensitivity. Our method discriminated between the L452R mutant and wild-type L452. In addition, HRM analysis distinguished the T478K mutant from the wild-type T478. Seven clinical samples containing the Delta variant were successfully identified as L452R/T478K mutants. These results indicate that this HRM-based genotyping method can identify the Delta variant. This simple method should contribute to rapid identification of the Delta variant and the prevention of infection spread.

Keywords: Delta variant; Genotyping method; High-resolution melting; L452R mutation; SARS-CoV-2; T478K mutation; ​.

MeSH terms

  • Biological Assay / methods*
  • COVID-19 / genetics*
  • Genetic Variation
  • Genotyping Techniques
  • Humans
  • Mutation*
  • Polymerase Chain Reaction / methods*
  • SARS-CoV-2 / genetics*
  • Transition Temperature