2019 ncov viewer

Allplex SARS-CoV-2 Variants I Assay from Seegene Inc was used according to the manufacturer protocol to perform rRT-PCR. Briefly, Extracted RNA (5 µl) was transferred to 96 well PCR plate containing 15 µll of the master mix. Plates were then spun down at 2500 rpm for 5 s and analyzed on a CFX96 Touch Real-Time PCR from BioRad. Reverse Transcription reaction 1 cycle: 50 °C/20 min – 95 °C/15 min. PCR reaction 45 cycles: 94 °C/15 s – 58 °C/30 sec. Gene amplifications were analyzed by FAM (E484K mutation on S-Gene), HEX (RdRP), Cal Red 610 (N501Y mutation on S-Gene), Quasar 705 (69-70del on S-Gene), and Quasar 670 (Human Endo Internal control) fluorophores. Results were compiled and analyzed using the 2019-nCoV viewer from Seegene Inc according to the manufacturer’s instructions.

To evaluate viral replication in cell cultures for the mutational induction assay, the Allplex SARS-CoV-2 Extraction-Free system (Seegene Inc., Seoul, Korea) was used. It consists of a real-time qRT-PCR multiplex assay based on the use of TaqMan probes, which allows simultaneous detection of four target genes, namely E gene, RdRP/S gene and N gene. Sample preparation, reaction setup and analysis were performed accordingly to the manufacturer instructions⁵⁹ . Briefly, 15 µL of sample were diluted 1:4 in 45 µL of RNase-free water in a 96-well PCR plate and hence 5 µL of the dilution were transferred to another plate with 16 µL of PCR master mix, containing 5 µL of MOM (MuDT Oligo Mixture, with dNTPs, oligos, primers and TaqMan 5’ fluorophore/3’ Black Hole Quencher probes), 5 µL of enzymes, 5 µL of RNase-free water and 1 µL of internal control for every reaction. A positive and a negative control were included. The assay was run on a CFX96 real-time thermal cycler (Bio-Rad, Feldkirchen, Germany). The amplification process includes cDNA denaturation at 95 °C for 10 s, primers annealing at 60 °C for 15 s and elongation at 72 °C for 10 s (44 cycles). Fluorescent signals were acquired after every amplification cycle. Results analysis and targets quantification were performed with 2019-nCoV viewer from Seegene Inc.