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

• Published in: Journal of virological methods Vol. 314; p. 114678
• Main Authors: Aoki, Akira, Adachi, Hirokazu, Mori, Yoko, Ito, Miyabi, Sato, Katsuhiko, Kinoshita, Masayoshi, Kuriki, Masahiro, Okuda, Kenji, Sakakibara, Toru, Okamoto, Yoshinori, Jinno, Hideto
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2023; Published by Elsevier B.V
• Subjects: A23063T; Class 4 single nucleotide polymorphisms; COVID-19 - diagnosis; High-resolution melting; Humans; Mutation; N501Y; Polymerase Chain Reaction - methods; Protocols; Rapid identification; SARS-CoV-2; SARS-CoV-2 - genetics; Temperature; Class 4 single nucleotide polymorphisms; SARS-CoV-2; N501Y; Rapid identification; High-resolution melting; A23063T
• ISSN: 0166-0934; 1879-0984; 1879-0984
• DOI: 10.1016/j.jviromet.2023.114678

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. •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.