A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

Abstract

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high-resolution melting assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected during the Alpha, Delta and Omicron [BA.1] waves of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities.

Figure 1

High resolution melt profiles (derivative, dF/dT) of multiplex assays A–D: (A) Multiplex A, targeting S_del.156–157, S_K417N and N_D3L, (B) Multiplex B, targeting S_E484K, S_del.242–244 and S_P681H/R, (C) Multiplex C, targeting Orf1ab_del.3675–3677, S_del.242–244 and S_P681H/R, and (D) Multiplex D, targeting S_del.156–157, S_K417N, N_D3L and S_EPE (all mutations and primer sets as described in Table 1). Data shows evaluation in vitro cultured SARS-CoV-2 strains, where the variant type had been determined by WGS, as follows: 19A (green line), Alpha VOC (purple line) SARS-CoV-2/human/GBR/FASTER_372/2021, Beta VOC (red line) hCoV-19/South Africa/KRISP-EC-K005321/2020, Gamma VOC (black line) hCoV-19/Japan/TY7-503/2021, Delta VOC (blue line) SARS-CoV-2/human/GBR/Liv_273/2021 and Omicron VOC RNA derived from a clinical specimen where SARS-CoV-2 RNA was detected at Ct < 30 and with known complete genome sequence.

Figure 2

Melt temperature (Tm, °C) value distributions (with 95% confidence intervals) for clinical samples collected 15th–22nd January 2021 (Alpha, N = 30) or collected between 14 th and 25th June 2021 (Delta, N = 30) where SARS-CoV-2 RNA was detected at Ct < 30 and with known complete genome sequences. Data are shown for each primer set (as described in Table 1): (A) S_del.156–157, (B) S_del.242–244, (C) S_K417N, (D) S_E484K, (E) S_P681H/R (F) Orf1ab_del.3675–3677, and (G) N_D3L. Blue dotted lines represent the positivity thresholds (shown in Table 3), which when crossed indicates the presence of the mutation. Positivity thresholds were calculated by the addition or subtraction of two standard deviations surrounding the mean Tm, of samples confirmed negative for the mutation.

Figure 3

Melt temperature (Tm, °C) value distributions (with 95% confidence intervals) for clinical samples collected 15th-22nd January 2021 (Alpha, N = 10) or collected between 14th and 25th June 2021 (Delta, N = 10) or collected between December 2021–January 2022 (Omicron, N = 16) where SARS-CoV-2 RNA was detected at Ct < 30 and with known complete genome sequences.