Influenza Diagnosis by RT-PCR

We performed RNA extraction and RT-PCR according to the Influenza Diagnosis Manual 4th edition (National Institute of Infectious Diseases, 2019) [11 ]. In gargle samples, total RNA was isolated from 140 µL of a gargle sample using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). For nasopharyngeal swabs, total RNA was isolated from 140 µL of the extraction buffer using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed using the primers and probes listed in Additional file 1: Table S1, the One-Step PrimeScript™ RT-PCR Kit (TAKARA BIO, Shiga, Japan), and QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). In brief, 2 μL of extracted RNA was added to 10 μL of 2X One Step RT-PCR Buffer III, 0.4 μL each of 10 μM primers (Additional file 1: Table S1), 0.25 μL of 10.2 μM Taqman Probe, 0.4 μL of 50X ROX Reference Dye II, 0.4 μL of PrimeScript RT enzyme Mix II, 0.4 μL of TaKaRa Ex Taq HS, and 5.75 μL of RNase free water. The conditions consisted of 1 cycle of 5 min at 42 °C, 10 s at 95 °C and followed by 45 cycles of 5 s at 95 °C, 34 s at 55 °C for H1N1 or 58 °C for H3N2 and B. The result was analyzed using QuantStudio (Thermo Fisher Scientific, Massachusetts, USA), in which a cycle threshold value (Ct-value) < 40 was defined as a positive result.
If the results of TRCsatFLU were different from those of RT-PCR, the TRC and RT-PCR products were analyzed by sequencing according to the Influenza Diagnosis Manual 4th edition (National Institute of Infectious Diseases, 2019) [11 ]. In brief, positive samples were purified using QIAquick PCR Purification kit (Qiagen, Hilden, Germany). Sequencing employed the ABI Big Dye Terminator system (ThermoFisher Scientific, Waltham, MA, USA). It was performed at a contract sequencing facility (FASMAC Co., Ltd. Kanagawa, Japan). For each sequencing reaction, 50 ng template and 3.2 pmol primers (Additional file 1: Table S1) were used.