RT-PCR for Influenza Virus Subtyping

RT-PCR for the classification of the influenza virus subtypes was performed as described previously [11 (link)] with minor modifications. Briefly, viral RNA was extracted from sample specimens using a QIAamp^® Viral RNA Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed with a OneStep RT-PCR kit (QIAGEN) using individual primer sets for H1N1dpm, H1N1 seasonal influenza before 2009, or H3 (Supplementary Table S1). The reaction components for RT-PCR were prepared by mixing 2 μL of 5× QIAGEN OneStep RT-PCR Buffer, 0.4 μL of dNTP mix, 0.2 μL of forward primer (10 μM), 0.2 μL of reverse primer (10 μM), 0.32 μL of QIAGEN OneStep RT-PCR Enzyme Mix, 5.68 μL of Nuclease-free H₂O, and 1.2 μL of Viral RNA per reaction. The PCR conditions were 50 °C for 30 min and 94 °C for 3 min, followed by 45 cycles of 94 °C for 30 s, 54 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 7 min. The PCR products were subjected to electrophoresis on a 2% agarose gel. RNA extracted from each positive control virus was used as the standard RNA. The concentration of each sample was calculated by fitting the infection titer of each virus to create a standard curve.