Nucleic acid extraction and real‐time reverse transcription polymerase chain reaction (rRT‐PCR)
INFLUENZA A, INFLUENZA, DISEASE, VIRAL SHEDDING, INFLUENZA
Viral RNA was extracted from a 50 μL volume of each sample using the MagMax™‐96 Viral Isolation kit (Thermo Fisher Scientific, USA), according to the manufacturer's instructions. Subsequently, to detect wild‐type Influenza A and B viruses and vaccine viruses (LAIV‐A and B), one‐step real‐time reverse‐transcription PCR (rRT‐PCR) was performed using specific oligonucleotide primer sets and probes provided by the Influenza Division of the Centers of Disease Control and Prevention (CDC), Atlanta, Georgia through the International Resource Reagent (The 25 μL PCR reaction composed of 12.5 μL 2X Agpath‐ID™ One‐Step RT‐PCR buffer (Applied Biosystems™, USA), 0.5 μL of 10 μM forward and reverse primers (target gene‐specific), 0.5 μL of 5 μM target‐specific probe, 1.0 μL of 25X RT‐PCR enzyme mix from Agpath‐ID™ One Step RT‐PCR kit, 5 μL of sample RNA, and 5.0 μL nuclease‐free water. The amplification process was performed in a StepOnePlus™ Real‐Time PCR System or an ABI 7500 Real‐Time PCR System (Applied Biosystems™, USA) according to the CDC protocol: 50°C for 30 min (reverse transcription) and 95°C for 10 min (DNA polymerase activation), followed by 45 cycles of 95 °C for 15 s (denaturation) and 55°C for 60 s (annealing and extension). The cycle threshold (Ct) value of each run reaction was normalized to the mean Ct of the pooled influenza positive control (CDC, USA) across all runs. Results were interpreted as per the protocol of CDC, USA.We expressed nasal shedding proportion among those who received the vaccine or placebo on days 2 and 4 for each year. We also looked at viral shedding by influenza type and subtypes.