Viral RNA was purified from cell culture supernatant using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. Subsequently, the purified RNA was used as template to synthesize the first-strand cDNA, using the SuperScript™ First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instruction. Real-time PCR, using SYBR® Green dye-based PCR amplification and detection method, was performed to detect the cDNA.
We used the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), the forward primer N2F: TTA CAA ACA TTG GCC GCA AA, the reverse primer N2R: GCG CGA CAT TCC 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 step at 72 °C for 10 min [16 (link)]. RT-PCR was performed using the ABI-PRISM 7900HT Fast Real-Time instruments (Applied Biosystems, Waltham, MA, USA) by using optical-grade 96-well plates. Samples were run in duplicate in a total volume of 20 μL.
We used the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), the forward primer N2F: TTA CAA ACA TTG GCC GCA AA, the reverse primer N2R: GCG CGA CAT TCC 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 step at 72 °C for 10 min [16 (link)]. RT-PCR was performed using the ABI-PRISM 7900HT Fast Real-Time instruments (Applied Biosystems, Waltham, MA, USA) by using optical-grade 96-well plates. Samples were run in duplicate in a total volume of 20 μL.