Influenza A and RSV subtyping
Next, we subtyped all the Influenza A and RSV-positive samples using
Seegene Allplex™ Respiratory Panel 1 (Seegene Inc, Seoul, South Korea).
Allplex™ Respiratory Panel 1 is a multiplex assay for simultaneous
detection and differentiation of three Influenza A subtypes (Influenza
A-H1, Influenza A-H1pdm09, Influenza A-H3) and two RSV subtypes (RSV A
and RSV B). Viral RNA was extracted from all Influenza A positive and
RSV-positive specimens using Zymo quick DNA/RNA extraction kit (Zymo
Research, Irvine, USA) according to the manufacturer’s protocols. 8µl of
extracted RNA was added to 17µl of mastermix, and RT-qPCR was done on
Bio-Rad CFX96 (Bio-Rad Laboratories, USA). Amplification was performed
at 50°C for 20 mins, 95°C for 15 mins, and 44 cycles of 95°C for 10
secs, followed by fluorescence detection at 60°C for 1 min and 72°C for
10 secs. All runs were performed together with relevant controls to
ensure validity. The results were exported to Excel and interpreted
using Seegene Viewer (Seegene Inc, Seoul, South Korea).