SARS-CoV-2 Viral Load Quantification

Viral loads in the respiratory tract, evaluated as a copies of subgenomic (sg)E and sgN RNA in the sample, were measured as previously reported^{27 (link),53 (link)}. BAL fluid and nasal swabs were stored in RNAzol BD (Molecular Research Center) and PBS until use, respectively. Total RNA was extracted with the RNAzol BD column kit (Molecular Research Center). TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems), gene-specific primers (sgLeadSARSCoV2_F: 5′-CGATCTCTTGTAGATCTGTTCTC-3′, E_Sarbeco_R: 5′-ATATTGCAGCAGTACGCACACA-3′, wtN_R: 5′-GGTGAACCAAGACGCAGTAT-3′) and probes (E_Sarbeco_P: 5′-FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3′, wtN_P: 5′-FAM-TAACCAGAATGGAGAACGCAGTGGG-BHQ1-3′) were used for the RT-qPCR reaction in 384-well plates (Bio-Rad). The total volume of the reaction was 20 μL, and the sample volume was 3.33 μL. CFX384 Touch Real-Time PCR Detection System (Bio-Rad) was used for amplifications. The lower limit of quantification was 50 copies/reaction.