2.2. Nucleic acid extraction and real time RT‐PCR

Saliva specimens were resuspended in 2 ml of PBS, 150 µl was subjected to RNA extraction by a Patho Gene‐spin Viral DNA/RNA Extraction Kit (Intron Biotechnology Inc.). RNA extraction was performed according to the manufacturer's instructions. The SARS‐CoV‐2 virus in nasopharyngeal swab and saliva specimens was detected by the real‐time RT‐PCR technique via using a Roche Lightcycler‐96 device (Roche Diagnostic Systems). RT‐PCR was performed using a SARS‐CoV‐2 (2019‐nCoV) qPCR Detection Kit (Bioeksen R&D) that is targeting a SARS‐CoV‐2‐specific RNA dependent RNA polymerase (RdRp) gene fragment. The final PCR concentration was 20‐µl (10‐µl qPCR master mix, 5‐µl primer/probe set, and 5‐µl template). The nucleic acid amplification was performed with the following PCR steps: Reverse transcription stage (45°C, 15 min, and 1 cycle), initial activation stage (95°C, 3 min, and 1 cycle), and amplification stage (Denaturation: 95°C, 5 s, annealing, and extension: 55°C, 35 s, and 45 cycles). All samples were run in two replicates, together with a SARS‐Cov‐2 positive control, and negative control. For data analysis, the 2‐∆∆Ct method was used and a cycle threshold (CT) value less than 40 is interpreted as positive for SARS‐CoV‐2 RNA.