Quantifying Avian Influenza Virus RNA

Virus RNA was extracted from cell supernatant or quail oropharyngeal swabs and quantitated (52 (link)). Viral RNA was isolated using the MagMAX-96 AI/ND viral RNA isolation kit (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions. A one-step qPCR based on the avian influenza virus matrix gene as a surrogate of virus replication was carried out in a LightCycler 480 real-time PCR instrument (Roche Diagnostics, Rotkreuz, Switzerland) using a LightCycler 480 RNA master hydrolysis probe kit (Roche Life Science, Mannheim, Germany) in a final reaction volume of 20 µL. Each reaction mixture contained 1 μ LightCycler 480 probes master mix, 0.5 µM forward and reverse primers, 0.3 µM probe, and 5 µL of RNA. The qPCR cycling conditions ran at 61°C for 10 min and a denaturation step of 95°C for 30 s, followed by 45 cycles of amplification at 95°C for 10 s, 60°C for 20 s, and 72°C for 1 s, with a final cooling step at 40°C for 10 s. A standard curve was generated using 10-fold serial dilutions of a WF10 virus stock of known titer to correlate qPCR crossing point values with the amount of virus in each sample.