Duplex Real-Time RT-PCR for PLHV-3 and Influenza A

The real-time RT-PCR was carried out according to McMahon et al. [49 (link)]. All reactions were prepared in a volume of 16 μl using SensiFAST Probe No-ROX One-Step Kit (Meridian Bioscience, Cincinnati, OH, USA) plus 4 μl template RNA. Experiments were performed as duplex real-time RT-PCR, thus detecting PLHV-3 and an internal Influenza A-RNA control as described [35 (link)]. Real-time RT-PCR was performed with a qTOWER3 G qPCR cycler (Analytik Jena, Jena, Germany) applying a temperature-time profile that consists of a reverse transcriptase step of 30 minutes at 50°C, followed by an activation step of 10 minutes at 90°C and 45 cycles comprising a step of 30 seconds at 90°C, followed by a step of annealing and extension of 30 seconds at 59°C.