SARS-CoV-2 RNA Quantification by RT-PCR

Viral RNA was purified from 140 μL of cell culture supernatant using the QIAamp Viral RNA Mini Kit (QIAGEN), following the manufacturer's instructions. Subsequently, the purified RNA was used to perform the synthesis of the first-strand cDNA, using the SuperScript™ First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific), following the manufacturer's instruction. Real-time PCR, using SYBR® Green dye-based PCR amplification and detection method, was performed in order to detect the cDNA. We used the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) the forward primer N2F: TTA CAA ACA TTG GCC GCA AA, the reverse primer N2R: GCG CGA CAT TCC GAA GAA, and the following PCR conditions: 95°C for 2 min, 45 cycles of 95°C for 20 s, annealing at 55°C for 20 s and elongation at 72°C for 30 s, followed by a final elongation at 72°C for 10 min (Hirotsu et al., 2020 (link)). RT-PCR was performed using the ABI-PRISM 7900HT Fast Real-Time instruments (Applied Biosystems) by using optical-grade 96-well plates. Samples were run in duplicate in a total volume of 20 μL.