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).