2.4 Real-time RT-PCR
Total RNA was extracted from all clinical samples and checked for the presence of SARS-Cov-2 RNA by an E gene specific semi quantitative real-time RT-PCR assay (RT-qPCR) that detects a broad range of human and bat coronaviruses (Corman et al., 2019). For the detection of SARS-CoV-2 RNA by RT-qPCR, we used 4X TaqMan® Fast Virus one step RT-PCR kit (Life Tech., USA) according to manufacturer’s recommendations. For each RT-qPCR reaction, 0.4 µM of each E gene forward and reverse primers and 0.2 µM of probe were used. RT-qPCR runs were performed using a 7500 Fast Real-Time PCR System (Applied Biosystem) using the following cycle conditions: 50°C for 5 min, 95°C for 20 sec, and 40 cycles of 95°C for 3s followed by 60°C for 30s. RT-qPCR semi-quantitative results were calculated based on a gBlock (Integrated DNA Technologies, IDT) standard curve for SARS-CoV-2 E gene.