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

Akira Aoki; Hirokazu Adachi; Yoko Mori; Miyabi Ito; Katsuhiko Sato; Masayoshi Kinoshita; Masahiro Kuriki; Kenji Okuda; Toru Sakakibara; Yoshinori Okamoto; Hideto Jinno

doi:10.1016/j.jviromet.2023.114678

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

J Virol Methods. 2023 Apr:314:114678. doi: 10.1016/j.jviromet.2023.114678. Epub 2023 Jan 18.

Authors

Affiliations

¹ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan.
² Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan.
³ Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan; Kiyosu Health Center, 129 Haruhi, Furikata, Kiyosu, Aichi 452-0961, Japan.
⁴ Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan; Handa Health Center, 1-45-4 Deguchi-cho, Handa, Aichi 475-0903, Japan.
⁵ Aichi Prefectural Institute of Public Health, 7-6 Nagare, Tsuji-machi, Kita-ku, Nagoya 462-8576, Japan; Nishio Health Center, 12 Shimoda, Yorizumi-cho, Nishio, Aichi 445-0073, Japan.
⁶ Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan. Electronic address: jinno@meijo-u.ac.jp.

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.

Keywords: A23063T; Class 4 single nucleotide polymorphisms; High-resolution melting; N501Y; Rapid identification; SARS-CoV-2.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

COVID-19* / diagnosis
Humans
Mutation
Polymerase Chain Reaction / methods
SARS-CoV-2* / genetics
Temperature

Supplementary concepts

SARS-CoV-2 variants