Rapid detection of the SARS-CoV-2 omicron variants based on high-resolution melting curve analysis

Abstract

With the continuous spread of the SARS-CoV-2 globally, viral mutations have accumulated. As a result, SARS-CoV-2 became more contagious, and has a higher risk of immune escape and reinfection. To identify variants and have an awareness of the prevalence of these variants, this study selected four segments containing mutations on the S gene of the SARS-CoV-2. Then a rapid and convenient variants detection method was established using high-resolution melting(HRM) analysis combined with nested polymerase chain reaction(PCR). The total detection process takes about 5 h. Through comprehensive analysis of the results from the four reaction systems, the identification of seven important Omicron variants(BA.2, BA.2.75, BA.5.2, BF.7, BQ.1, XBB.1 and XBB.2) can be achieved, with significant differentiation in the melting curves of each variant group. The method established in this study was used to genotype positive specimens in COVID-19 nucleic acid testing, the overall concordance rate compared to whole genome sequencing results was 88.9%, and the positive concordance rate of each sublineage was greater than 80% and the negative concordance rate was greater than 94.4%. The detection of clinical specimens has demonstrated that the HRM analysis established in this study is an effective, rapid and convenient variant identification method, which can be used for monitoring SARS-CoV-2 variants and has important value in addressing public health issues caused by the ongoing mutations of the SARS-CoV-2.

Keywords: HRM Analysis; Omicron; SARS-CoV-2; Variant detection.

Fig. 1

Mutations combinations for detection of the SARS-CoV-2 Omicron variants.

Fig. 2

Schematic diagram of primer design for variant sites amplification: (A) Primer design for amino acid regions 210–213 and 252–257; (B) Primer design for amino acid region 440–460; (C) Primer design for amino acid position 1243.

Fig. 3

Melting curves of HRM detection for the I210 and V213 mutations in Omicron variants: (A) Normalized and Shifted Melting Curve; (B) Normalized and Temp-Shifted Difference Plot; (C) Melting Peaks.

Fig. 4

Melting curves of HRM detection for the G252, D253 and G257 mutations in Omicron variants: (A) Normalized and Shifted Melting Curve; (B) Normalized and Temp-Shifted Difference Plot; (C) Melting Peaks.

Fig. 5

Melting curves of HRM detection for the amino acid 440–460 region mutations in Omicron variants: (A) Normalized and Shifted Melting Curve; (B) Normalized and Temp-Shifted Difference Plot; (C) Melting Peaks.

Fig. 6

Melting curves of HRM detection for the C1243F mutation in Omicron variants: (A) Normalized and Shifted Melting Curve; (B) Normalized and Temp-Shifted Difference Plot; (C) Melting Peaks.