Multiplex HRM Assays for SARS-CoV-2 VOCs

Initially, three multiplex HRM assays, each containing three different primer pairs were developed and evaluated in this study. These assays identified combinations of variant-defining mutations for SARS-CoV-2 VOCs, with a focus on differentiation of Alpha from 19A, and later Delta from Alpha, to understand the epidemiology of these variants at the time in the UK.
Each assay was performed using 2.5 μL of RNA template and in 12.5 μl final reaction volumes, using the SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase kit (ThermoFisher, USA), with final reagent quantities as follows: 1X reaction mix, 0.25 μL of SuperScript™ III RT/Platinum™ Taq Mix, 1X EvaGreen® dye (Biotium, USA), and primers added to their optimised concentration (Table 2).Table 2

Optimised final reaction primer concentrations for primer set in each multiplex assay, A-D. Primer set names as described in Table 1.

Primer set	Final forward primer concentration (nM)	Final reverse primer concentration (nM)
Multiplex A
S_del.156–157	100	150
S_K417N	150	225
N_D3L	600	900
Multiplex B
S_E484K	300	450
S_del.242–244	150	225
S_P681H/R	150	225
Multiplex C
Orf1ab_del.3675–3677	200	300
S_del.242–244	200	300
N_D3L	175	262.5
Multiplex D
S_del.156–157	100	100
S_K417N	150	150
N_D3L	600	600
S_EPE	250	250

Reactions were performed using the RGQ 6000 5-plex HRM platform (Qiagen, Germany) with the following thermal cycle profile: reverse transcription at 50 °C for 15 min, initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 56 °C for 30 s and 72 °C for 20 s. HRM was then performed, melting from 73 °C to 85 °C, acquiring data to the HRM channel in 0.1 °C increments, with a 2 s stabilisation between each step. For each assay a reference control of 19A strain SARS-CoV-2 RNA, confirmed by WGS to be negative for all mutations of interest, and a no template negative control were included.