Allplex sars cov 2 assay

A total of 20 SARS-CoV-2 strains, including one wild-type and 19 mutants, were obtained from the Korea Disease Control and Prevention Agency (KDCA) (Table S1). For clinical testing, we used clinical SARS-CoV-2 NP samples (n = 92) and clinically normal NP samples (negative control group, n = 100) collected from SARS-CoV-2-infected (from June 2022 to July 2022) and non-infected (from February 2018 to November 2019) patients at Korea University Guro Hospital. All clinical samples were confirmed using the Allplex^TM SARS-CoV-2 assay (Seegene Inc., Seoul, Republic of Korea). For cross-reactivity testing, a total of 42 clinical respiratory samples (9 of coronavirus (229E, NL63, and OC43), 3 of influenza virus A/H1N1, 3 of influenza virus A/H3N2, 3 of influenza virus B, 3 of respiratory syncytial virus (RSV) A, 3 of RSV B, 3 of adenovirus (AdV), 3 of parainfluenza virus (PIV), 3 of human bocavirus (HboV), 3 of human enterovirus (HEV), 3 of human rhinovirus (HRV), and 3 of metapneumovirus (MPV)) were collected from Korea University Guro Hospital and confirmed by PCR using the Anyplex^TM II RV16 detection kit (Seegene Inc., Seoul, Republic of Korea). This study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Korea University Guro Hospital (approval number: 2019GR0055).

Microbiological analysis for the etiological diagnosis of pneumonia included blood-culture (BD BACTEC™ blood culture systems), urine antigen test for Streptococcus pneumoniae and Legionella pneumophila detection (Sofia FIA^® Quidel Corporation, San Diego, CA, USA), and qRT-PCR targeting viral respiratory pathogens (Allplex™ Respiratory Panels 1, 2 and 3, Seegene, South Korea). For other less frequent bacterial pathogens causing pneumonia (Mycoplasma pneumoniae and Chlamydophila pneumoniae) a PCR of a pharyngeal swab was performed according to manufactures instructions (CerTest Biotec SL, Spain).
In patients with diagnosis of SARS-CoV-2 pneumonia, diagnosis of SARS-CoV-2 had been commonly done some days before admission using commercial RT-PCR (Allplex SARS-CoV-2 assay, Seegene, South Korea) on nasopharyngeal swabs. Alpha (lineage B.1.1.7) was the predominant SARS-CoV-2 variant present when samples of these patients were collected.

In this study, two rapid nucleic acid extraction reagents were used: Pi-Lise Nucleic Acid Extraction Reagent, by Pi-Biotech Genética Avançada Ltda (Santos Dumont, MG, Brazil), and QuickExtract^TM DNA Extraction Solution, by Lucigen Corporation (Middleton, WI, USA). Once thawed, aliquots were stored at −20 °C to avoid >3 freeze–thaw cycles.
To perform the RT-qPCR assays, a multiplex kit (Allplex SARS-CoV-2 Assay, by Seegene Inc., Seoul, Republic of Korea), and a singleplex set of reagents [iTaq Universal Probes One-Step Kit, by Bio-Rad Laboratories (Hercules, CA, USA),were used, together with primers and controls 2019-nCoV RUO by IDT Integrated DNA Technologies (Coralville, IA, USA)]. The analyses were run in two distinct equipment: QuantStudio^™ 3 and 7500Fast Real-Time PCR Systems, by Applied Biosystems (Foster, CA, USA).

All swab samples underwent nucleic acid extraction and real-time polymerase chain reaction (PCR) for SARS-CoV-2 with commercial extraction and diagnostic kits routinely used in each site. All kits have received approval from the Medical Device Authority, Malaysia. For extraction, KLGH, HT and SH used RNA Isolation Kit with the KingFisher Duo Prime Purification System (Thermo Fisher Scientific, USA), Viral RNA Isolation Kit with the Liferiver EX3600 (Liferiver Biotech, China), QIAsymphony DSP Virus/Pathogen Mini Kit (Qiagen, Germany), and/or the Magcore Plus II Viral Nucleic Acid Extraction Kit (RBS Bioscience, Taiwan); PCR was performed with Real-Q 2019-nCoV Detection Kit (BioSewoom, South Korea), LyteStar 2019-nCoV RT-PCR Kit (ADT Biotech, Malaysia) and Allplex SARS-CoV-2 Assay (Seegene, South Korea). UMMC used either the GENTi Advanced DNA/RNA Extraction Kit (GeneAll, Korea) and Allplex SARS-CoV-2 Assay or the fully automated cobas SARS-CoV-2 Test on the cobas 6,800 system (Roche, USA). In addition, samples were tested for influenza A and B viruses and respiratory syncytial virus (RSV) using the Xpert Xpress Flu/RSV kits and GeneXpert rapid real-time PCR system (Cepheid, USA). Assays were performed and results were analyzed following manufacturers’ instructions. All PCR tests were performed within 48 h of sample collection.