Nucleic acids extraction and virus detection.
Nucleic acids were extracted and purificated by using the automated nucleic acid purifier Magnapure (Roche Diagnostics SL, Swizertland) following manufacturer´s instructions. Extracted nucleic acids were resuspended in a final volume of 70 µl.
RSV genome was detected and quantificated by a multiplex real time reverse transcription polymerase chain reaction rt-RT-PCR for Influenzavirus A and B and RSV using type-specific primer pairs and MGB probes (Table 1) and the TaqMan Fast 1-Step Master Mix (Life technologies, CA). RT-PCR was performed with 5 µl of extracted nucleic acids in a final volume of 10 µl as follows: 50º/10’, 95º/7’, 45 cycles of 95º/5” and 60º/33”.
In addition, the human ß-globin gene was quantified in each sample in order to evaluate sample quality and to calculate normalized viral load in copies/103 cells.
Table 1. Primers and probe used for detection of Influenza A (IA), Influenza B (IB) and RSV (types A and B) and sequencing