RNA extraction and detection of SARS-CoV-2 RNA by standard RT-qPCR
RNA extraction was done under BSL-2 on 410 µl of the VTM of both swabs using the QIAamp® Viral RNA mini kit (cat. no. 52906, Qiagen) according to the manufacturer’s recommendation. During the extraction, RNase-free DNase set (cat no. 79254, Qiagen) was used to treat the RNA samples to eliminate the possibility of genomic DNA contamination. RNA quality and quantity were determined with the Nanodrop S1000 spectrophotometer (Thermo fisher Scientific). A one-step RT-qPCR was done on extracted RNA using real-time PCR kit (Primerdesign Ltd, Ref: Z-Path-COMD-19-CE, UK) in Stratagene Mx3000P qPCR System (Agilent). This assay targets RNA-dependent RNA polymerase (RdRP ) gene within SARS-CoV-2. The 20 µl reaction mix formed of 10 µl 2X RT-qPCR Master Mix, 2 µl of COVID-19 Primer & Probe and 8 µl sample extract. A positive control template and negative amplification control with nuclease-free water were included in each run. In the one-step protocol, the reverse transcription (complementary DNA; cDNA; formation) was done by heating the mix at 55 °C for 10 min. and the cDNA was heated at 95 °C for 2 min. (initial denaturation) followed by 45 cycles, each consists of denaturation at 95 °C for 10 sec., annealing and extension at 60 °C for 1 min. The cycle threshold (Ct ) values were recorded for each sample. The analyzed samples were considered negative if they have a Ct value ≥40 or no Ct values were reported. For positive samples, SARS-CoV-2 RNA content was categorized according to the Ct values into high (Ct < 29), moderate (Ct = 29-36) and low (Ct = 37-39).