Quantification of viral copies by RT-PCR

Viral RNA copies in the lungs of PR8-infected mice were evaluated with a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay by targeting the conserved matrix gene of influenza virus.⁴⁵ A serially diluted recombinant plasmid (pET-28b(+)/M1) containing the target gene was used as a standard. The lungs from influenza virus-infected mice were harvested at the indicated time and homogenized in PBS. Total RNA was extracted with an RNeasy plus mini kit (QIAGEN) following the manufacturer’s instructions. Using the QuantiNova Probe RT-PCR Kit (QIAGEN), one-step qRT-PCR was applied to detect viral RNA with primers (forward primer, 5′-CTTCTAACCGAGGTCGAAACGTA-3′; reverse primer, 5′-GGTGACAGGATTGGTCTTGTCTTTA-3′) and a TaqMan probe (5′[Fam]-TCAGGCC CCTCAAAGCCGAG-[BHQ-1]3′). The cycling conditions on the ABI Prism 7900HT Fast Real-Time PCR System consisted of 45 °C for 10 min and 95 °C for 5 min, followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s.