RNA extraction and RT-qPCR:
Sample drawing was performed by trained healthcare personnel via
nasopharyngeal swab. Immediately after collection, viral RNA was
extracted using the Indimag Pathogen kit (Indical Bioscience GmbH,
Germany) and tested for SARS-CoV-2 by RT-qPCR using the Bio-Rad CFX96
system (Roche, Switzerland) with a LightMix Modular Assay kit in
accordance with the modified Charité guidelines . 10 µl of extracted RNA
were added into 15µl reaction mixture (mastermix). Each 15µl mastermix
contained 12.5 µl buffer solution, 0.25 µl enzyme mix, 1.75 µl of
nuclease-free water and 0.5 µl primer probe wHCoV (ORF1ab: E-Gene,
occasionally N-Gene/Rd-Gene). Reactions were incubated at 55°C for 5
minutes and 95°C for 5 minutes in order to conduct reverse transcription
of viral RNA, sample denaturation and enzyme activation. These steps
were followed by PCR-amplification with 45 cycles at 95°C for 5s, 60°C
for 15s and 72°C for 15s. Cooling was implemented at 10°C for 30s.
Results were interpreted based on the Second derivative maximum (SDM)
method (Tichopad et al., 2003). Positive results were confirmed by RdRp-
and N-gene (Corman et al., 2020) RT-qPCR. Samples with an initial Cq
value lower than or equal to 37 were assigned to repeated testing
including extraction. A Cq value higher than 40 was defined negative.