Cfx maestro 2

The method requires two parallel RT-PCR reactions carried out in separate wells. Reaction ‘A’ is designed to detect any SARS-CoV-2 sequence other than the Delta variant, i.e., non-Delta. Reaction ‘B’ is designed to detect only the Delta variant. The primers used for Reaction ‘A’ and Reaction ‘B’ are detailed in Table 1. The amplicons generated in both reactions are detected by a common fluorescently labelled hydrolysis probe (Table 1). Primers and probes were synthesized by Integrated DNA Technologies IDT, Belgium. Five μL RNA template was used in a 20 μL reaction containing 5 μL of 4x TaqMan Fast virus 1-step mastermix (Applied Biosystems), primers at 400 nM and probe at 250 nM. Thermal cycling was performed in a Bio-Rad CFX real-time PCR system with reverse transcription at 54 °C for 10 min, followed by 94 °C for 3 min, then 40 cycles of 94 °C for 15 s and 58 °C for 30 s. Data were processed using Bio-Rad CFX maestro 2.0 software with baseline subtracted curve fit, fluorescence drift correction, automatically calculated baseline cycles and manual threshold settings. Each run included IC19 RNA and Delta #395 RNA, both diluted a million-fold, as positive controls. Several no-template nuclease-free water negative controls were also included in each run.