Design of primers and PCR protocol
Primers were designed after alignment of 311 sequences from clades 2 and
3 (published up to 29th June 2022) and sequences
published by Isidro and colleagues13, using MAFFT 7
online software14. Twelve amplicons
(~30,000 bp) were realized to detect 26/35 of the
characteristic mutations which differentiated 2022 outbreak clade 3 and
pre-epidemic clade 3 2018_UK_P2 (GenBank: MT903344.1)13. The nine uncovered mutations were all synonymous
or non-coding.
Amplification was realised using 3µL of RNA, 500nM of each primer
(Supplementary Table S1 ) and the Master Mix for PCR Platinum™
SuperFi II kit (Thermo Fisher) (12.5µL of 2X reaction mix) in a 25µL
final volume. The thermal profile used was 2 min at 98°C, 40 cycles
consisting of denaturation at 98°C, 10 s; hybridisation at 56°C, 10 s;
and elongation 68°C, 2 min 30 s ending with final elongation 68°C 5 min.
PCR products were pooled in equimolar proportions after purification
using Nucleofast PCR Plate kit (Macherey Nagel).