Viral load by quantitative real-time PCR analysis
In BSL3 experiments conducted in the BSL3 facility at the Icahn School of Medicine at Mount Sinai, Genomic viral RNA was extracted from supernatants using TRIzol reagent according to the manufacturer’s instructions (Thermo Fisher). RNA was reverse transcribed into cDNA using oligo d(T) primers and SuperScript II Reverse Transcriptase (Thermo Fisher). Quantitative real-time PCR was performed on a LightCycler 480 Instrument II (Roche) using KAPA SYBR FAST qPCR Master Mix Kit (KAPA biosystems) and primers specific for the SARS-CoV-2 nsp14 transcript as described previously (In BSL3 experiments conducted in the BSL3 facility at the Sheba Medical Center, Total nucleic acids were extracted from all samples using MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche) according to the manufacturer protocol. Extracted RNA was transferred to 96 well PCR plate containing 20 µl of TaqPath 1-step Multiplex Master Mix No ROX (Applied Bioscience, Cat number: A28523). This was followed by a one-step RT-PCR (TaqPath COVID-19 assay kit; Thermo-Fisher). Thereafter, the plate was sealed with MicroAmp clear adhesive strip (Applied Bioscience, Cat number: 4306311). The plate was loaded onto a QuantStudio 5 Real-Time PCR System (Applied Bioscience, Cat number: AB-A28574) and the following amplification program was used: 25 °C for 2 min, X1 cycle 53 °C for 10 min, X1 cycle 95 °C for 2 min, X1 cycle 95 °C for 3 s, followed by 60 °C for 30 s, X40 cycles Ct threshold values were presented using the following values/parameters: MS2-15,000; by cycle 37; S gene- 20,000 by cycle 37; Orf1ab- 20,000 by cycle 37; Ngene- 20,000 by cycle 37. Samples that passed the Threshold is a Ct value >37 were re-tested or considered weak positive. The viral load for each sample was determined using genomic viral RNA purified from viral stocks to generate a standard curve. Error bars indicate the standard error from three biological replicates.