A modified high-resolution melting-based assay (HRM) to identify the SARS-CoV-2 N501Y variant

Highlights

• Method to identify the SARS-CoV-2 N501Y by conventional and modified HRM assays.

• Conventional HRM can detect 10³–10⁶ copies/mL.

• Modified HRM can detect 10⁵–10¹⁰ copies/mL.

• Combination of two HRM assays with 93.3% sensitivity and 100% specificity.

Abstract

High-resolution melting (HRM) analysis is a PCR–based method that can be used as a screening assay to identify SARS-CoV-2 variants. However, conventional HRM assays hardly detect slight melting temperature differences at the A–T to T–A transversion. As the N501Y substitution results from A–T to T–A transversion in A23063, few or no studies have shown that a conventional HRM assay can identify N501Y variants. This study successfully developed an HRM assay for identifying the N501Y mutation. Two HRM assays were used in the N501 site because the discrimination results were affected by the virus copy numbers. One is a conventional HRM assay (detectable at 10³–10⁶ copies/mL) and the other is a modified HRM assay by adding the wild-type fragment (detectable at 10⁵–10¹⁰ copies/mL). Using viral RNAs from cultured variants (Alpha, Beta, and Gamma), a modified HRM assay correctly identified three N501Y variants because of high-copy-number RNAs in those viral samples. The sensitivity and specificity of the N501Y assay were 93.3% and 100%, respectively, based on 209 clinical samples (105 for N501; 104 for N501Y). These results suggest that our HRM-based assay is a powerful tool for rapidly identifying various SARS-CoV-2 variants.