Quantstudio 5

RT‐qPCR reactions were optimized on a Mx3005P (Agilent Technologies), QuantStudio 5 (Agilent Technologies) and AriaMx (Agilent Technologies) real‐time PCR detection systems using SOLIScript^® 1‐step CoV Kit (Cat. No. 08‐65‐00250; SOLIS BioDyne, Tartu, Estonia). For all genes, the reaction mixture was prepared according to the manufacturer´s recommendations comprised of 4 µl of 5× One‐step Probe CoV Mix (ROX), 0.5 µl of 40× One‐step SOLIScript^® CoV Mix, 2 µl of primers/probe mix, 8.5 µl of PCR water and 5 µl of sample in a 20 µl total volume. One‐step RT‐qPCR assays were conducted with the following cycling conditions: 55°C for 10 min for reverse transcription, 95°C for 10 min and 45 cycles of 95°C for 15 s and 60°C for 30 s. The sequences of primers and probes are shown in Table 1 and Table S1.

RT‐qPCR reactions were optimized on a CFX96 (Bio‐Rad), QuantStudio 5 (Agilent Technologies), Mx3005P (Agilent Technologies) and AriaMx (Agilent Technologies) real time PCR detection systems using Brilliant III Ultra‐Fast QRT‐PCR Master Mix (Cat. No. 600884; Agilent Technologies). For E, RdRP and RNase P genes, the reaction mixture was prepared according to the manufacturer´s recommendations comprised of 10 µl of 2× Brilliant III Ultra‐Fast QRT‐PCR Master Mix, 0.3 µl of 2 µM ROX, 0.2 µl of 100 mM DTT, 1 µl of RT/RNase Block, 2 µl of primers/probe mix, 1.5 µl of PCR water and 5 µl of sample in a 20 µl total volume. One‐step RT‐qPCR assays were conducted with the following cycling conditions: 50°C for 30 min for reverse transcription, 95°C for 3 min and 45 cycles of 95°C for 5 s and 60°C for 20 s. The sequences of primers and probes are shown in Table 1 and Table S1.