2.3 Identification of SARS-CoV-2 and viral coinfections by RT-qPCR and qPCR
The viruses were evaluated according to the Guidelines for Laboratory Surveillance of Influenza and Other Respiratory Viruses from the Institute of Epidemiological Diagnosis and Reference (Instituto de Diagnóstico y Referencia Epidemiológicos )10 and following the method of Corman et al.11. The SuperScript™ III Platinum® One-Step RT-qPCR System Kit (Invitrogen, Carlsbad, California, USA, Catalogue: 12574035) was used for the amplification of the viral genetic material in a 7500 Fast Real-Time PCR System (Applied Biosystems®, Foster City, California, USA). The viruses were evaluated in uniplex reactions with the following reaction mixture: 12.5 μl of 2x reaction mixture, 0.5 μl of each primer and probe, 0.5 μl of enzyme, 5.5 μl of RNase-free water, and 5 μl of total nucleic acid. The following thermocycling conditions were used: one cycle of 50 °C for 15 min and 95 °C for 2 min, followed by 45 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds (SARS-CoV-2) or 55 °C for 1 min (other viruses). The sequences of the primers and probes, as well as the working concentration of each, are given in Table 1.