Detection of other respiratory pathogens
All the positive SARS-CoV-2 samples were then analyzed for co-infection
with other respiratory pathogens using Seegene Allplex™ Respiratory
Panels 2, 3, and 4 (Seegene Inc, Seoul, South Korea). These panels are
multiplex kits for the identification of 12 viral and 7 bacterial
pathogens. Respiratory panel 2 identifies Adenovirus, Enterovirus,
Metapneumovirus and Parainfluenza virus types 1-4, whereas respiratory
panel 3 is for identification of human bocavirus 1/2/3/4, human
coronaviruses 229E, NL63, OC43 and human rhinovirus. Respiratory panel 4
is for identification of respiratory bacterial pathogensBordetella parapertussis, Bordetella pertussis,
Chlamydophila pneumoniae, Haemophilus influenzae, Legionella
pneumophila, Mycoplasma pneumoniae and Streptococcuspneumoniae. Nucleic acids were extracted from the specimens using
Zymo quick DNA/RNA extraction kit (Zymo Research, Irvine, USA) according
to the manufacturer’s protocols. 8µl of extracted nucleic acids was
added to 17µl of mastermix, and RT-qPCR was performed on Bio-Rad CFX96
(Bio-Rad Laboratories, USA). Amplification was performed at 50°C for 20
mins, 95°C for 15 mins, 44 cycles of 95°C for 10 secs, followed by
fluorescence detection at 60°C for 1 min and 72°C for 10 secs. The
cycling conditions were the same for all the panels. 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).