Multiplex RT-PCR for Respiratory Viruses

RT-PCR for Influenza-A, Influenza-B, RSV-A, RSV-B and Adenovirus virus were performed using a BHQ quencher probe at 125 nM and 250 nM of primers in a final volume of 20 μl. Five microliters of the extracted RNA was combined with 15 μl of the master mix. RT-PCR conditions was 60 cycles of 15 s at 95 °C and 45 s at 60 °C. This process was performed in Light Cycler® 2.0 Instrument and the data was analyzed with the LightCycler® Software 4.1 (Roche Diagnostic, Deutschland-Mannheim, Germany). The primers and the probe for Influenza A and B were described by Selvaraju et al., 2010 [23 (link)], for RSV-A and RSV-B were described by Liu W. et al., 2016 [24 (link)] and for Adenovirus was described by Heim et al., 2003 [25 (link)].
For he case of Parainfluenza 1 virus, Parainfluenza 2 virus and Parainfluenza 3, the primers and conditions for RT-PCR were described by Coiras et al., 004 [26 (link)].