Biorad cfx96 thermal cycler

RT-qPCR reactions for the two reference assays and the “designed in this study” Duo RT-qPCR were performed with SuperScript^® III Platinum^® One-Step RT-qPCR Kit with ROX (#11732-088, Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) on a BioRad CFX96^TM thermal cycler, software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA). A 5 μL volume of RNA was added to 20 μL of mix containing 12.5 μL of 2X Reaction Mix, 0.5 μL of Superscript III RT/Platinum Taq Mix and primers and probe at the concentrations described in Table 1. Cycling conditions were: 50 °C for 30 min; 95 °C for 2 min; 45 cycles of 95 °C for 15 s and 60 °C for 45 s. All probes were labeled with the same dye (FAM). There are no modifications for neither the sequence nor the concentrations of the primers and probes. The only difference is that the quencher of the probe described by Panning et al. has been modified for TAMRA, instead of BHQ in the original article. The reason for this is the need to have the same quencher for the probes of the two assays included in the Duo test.

RT-qPCR reactions for the three monoplex assays and the “designed in this study” Trio TOSV RT-qPCR assay were performed with a SuperScript^® III Platinum^® One-Step RT-qPCR Kit with ROX (#11732-088, Invitrogen—Thermo Fisher Scientific, Waltham, MA, USA) on a BioRad CFX96TM thermal cycler, software version 3.1 (Bio-Rad Laboratories, Hercules, CA, USA). A volume of 5 µL of RNA was added to 20 µL of mix containing 12.5 µL of 2× Reaction Mix, 0.5 µL of Superscript III RT/Platinum Taq Mix, and primers and probes at the concentrations described in Table 1. Cycling conditions were: 50 °C for 15 min; 95 °C for 2 min; 45 cycles of 95 °C for 15 s; 60 °C for 45 s. All probes were labeled with the same dye (FAM). There were no modifications for neither the sequence nor the concentrations of the primers and probes of the three monoplex assays when combined in the same reaction tube. The only difference is that the quencher of the probe described by Pérez-Ruiz et al. has been modified to TAMRA instead of Dabcyl that was used in the original article. The reason for this is the need to have the same quencher for the probes of the three assays included in the Trio test.