Real-Time RT-qPCR for SARS-CoV-2 Quantification

The real-time RT-qPCR protocol was adapted from the one used by Corman et al. [28 (link)]. Briefly, a 20 μL reaction was prepared containing 5 μL RNA, 400 nM primers, 200 nM probe, and 10 μL of 2 × reaction buffer provided with the iTaq universal Probe One-Step Kit (BioRad, Hercules, CA, USA). Oligonucleotides and probes targeting the E viral gene and human RNase P gene were synthesized and provided by LGC Biosearch Technologies (Petaluma, CA, USA). Thermal cycling was performed at 55 °C for 10 min for reverse transcription, followed by 95 °C for 3 min, and 45 cycles of 95 °C for 15 s and 58 °C for 30 s. To quantify the viral load, Cqs from the BioRad CFX96 system targeting the E gene were converted to viral load using a plasmid containing the sequence of SARS-CoV-2 genes E and RdRp, kindly provided by Jaime Castellanos’ Virology Laboratory, Universidad del Rosario, Colombia.