RSV characterization
During 2021 and 2022, 116 RSV positive samples under 25 Ct were randomly chosen and genotyped by Sanger sequencing method. A fragment of the G gene was amplified by a nested-PCR. The first round of amplification was carried out using the OneStep RT-PCR kit (EURx, Poland) and specific outer primers (Table 3). Five microliters of extracted viral genome were added to 20 µl of a RT-PCR mixture according to the manufacturer’s instructions. Amplification was performed using a GeneAmp PCR system 9600 thermal cycler (Applied Byosistems, USA) with the following conditions: 48º/45’, 95º/2’, 40 cycles of 95º/30”-55º/30”-72º/30”, and 72º/10’.
To increase sensibility, a second round of amplification was carried out. Three microliters of the previous amplification reaction were added to 22 μl of a PCR mixture containing 0.5 pmol of specific inner primers (Table 1), 50 μM of each dNTPs (Gibco BRL, Carlsbad, CA, USA), and 1 U of Taq DNA polymerase (Gibco BRL) in PCR buffer (1x) supplied by manufacturer. The amplification protocol was as follows: 95º/5’, 40 cycles of 95º/30”-55º/30”-72º/30”, and 72º/10’.
PCR products were analyzed by agarose gel electrophoresis, extracted by using Montage DNA Gel Extraction Kit (Millipore, USA) and sequenced with Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) supplemented with inner primers using an ABI 3130 genetic analyzer (Applied Biosystems, USA).
Demographic data (age and sex) from those patients are in File S1.