Isolation of viral RNA was carried out using a commercially available RNA isolation kit (Viral DNA/RNA Isolation Kit, A&A Biotechnology, Poland) according to the protocol provided by the manufacturer. Isolated RNA was subjected to reverse transcription (RT) and quantitative real-time PCR (RT-qPCR) using the GoTaq® Probe 1-Step RT-qPCR System Protocol kit (Promega, Madison, WI, USA). Due to the well-known ability of highly charged polymers to affect the RNA isolation process, the supernatants were diluted 1000-fold prior to isolation [28 (link)].
The RT-qPCR reaction was carried out using 3 μL of isolated viral RNA, which was reverse transcribed and amplified in a 10 μL reaction containing 1 × GoScript ™ RT Mix for 1-Step RT-qPCR, 1× GoTaq Probe qPCR Master Mix with dUTP, 300 nM specific probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) (5′ FAM–CGG CAT ACA GCA TCA GGT GCA TAG GAG-TAMRA-3′), and 450 nM of each primer (5′ TTG GTC ATG ATA CTG CTG ATT GC 3′ and 5′ CCT TCC ACA AAG TCC CTA TTG C 3′). The reaction was carried out in a thermal cycler (CFX96 Touch Real-197 Time PCR Detection System, Bio-Rad) under the following conditions: 45 °C 15 min (reverse transcription), 95 °C 2 min, then 40 cycles of 15 s at 95 °C and 30 s at 60 °C. Appropriate standards were prepared to evaluate the number of viral RNA molecules in the sample.
The RT-qPCR reaction was carried out using 3 μL of isolated viral RNA, which was reverse transcribed and amplified in a 10 μL reaction containing 1 × GoScript ™ RT Mix for 1-Step RT-qPCR, 1× GoTaq Probe qPCR Master Mix with dUTP, 300 nM specific probe labeled with 6-carboxyfluorescein (FAM) and 6-carboxytetramethylrhodamine (TAMRA) (5′ FAM–CGG CAT ACA GCA TCA GGT GCA TAG GAG-TAMRA-3′), and 450 nM of each primer (5′ TTG GTC ATG ATA CTG CTG ATT GC 3′ and 5′ CCT TCC ACA AAG TCC CTA TTG C 3′). The reaction was carried out in a thermal cycler (CFX96 Touch Real-197 Time PCR Detection System, Bio-Rad) under the following conditions: 45 °C 15 min (reverse transcription), 95 °C 2 min, then 40 cycles of 15 s at 95 °C and 30 s at 60 °C. Appropriate standards were prepared to evaluate the number of viral RNA molecules in the sample.
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