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.