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).