Quantitative RT-PCR for SARS-CoV-2 and Influenza

Total RNA was extracted from organs by Pure link RNA kit (Thermo Fisher Scientific). RT-qPCR was performed with viral gene specific primers^{26 (link),27 (link)} using the One Step PrimeScript™ III RT-qPCR Mix (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The N gene of SARS-CoV-2 or the M gene of IAV was amplified using the following paired primers and probes; forward primer 5′-AAATTTTGGGGACCAGGAAC-3′, reverse primer 5′-TGGCAGCTGTGTAGGTCAAC-3′, probe 5′-(FAM)ATGTCGCGCATTGGCATGGA(BHQ)-3′ (SARS-CoV-2) or forward primer 5′-CCMAGGTCGAAACGTAYGTTCTCTCTATC-3′, reverse primer 5′-TGACAGRATYGGTCTTGTCTTTAGCCAYTCCA-3′, probe 5′-(FAM)ATYTCGGCTTTGAGGGGGCCTG(MGB)-3′ (IAV), respectively. The following template cDNA were used for standard; 5′-GCCAGTGAATTGTAATACGACTCACTATAGGGCGAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAA-3′ (SARS-CoV-2) and 5′-AGTCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCACCTCTGACTA-3′ (IAV).