Lightcycler 480 instrument 2 platform

For the detection of SARS-CoV-2 RNA, quantitative RT-PCR was conducted on the LightCycler 480 Instrument II platform using the LightCycler Multiplex RNA Virus Master kit (Hoffmann-La Roche, Basel, Switzerland). The applied primers and probes were specific for the nucleocapsid 1 (N1) gene of the virus (2019-nCoV-N1-F, GAC CCC AAA ATC AGC GAA AT; 2019-nCoV-N1-R: TCT GGT TAC TGC CAG TTG AAT CTG; 2019-nCoV-N1-P: FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1) (CDC, Atlanta, https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). Virus copy number was calculated based on the standard curve of the commercially available copy control (EURM-019) obtained from the European Commission Joint Research Center, Geel, measured with an automatic threshold. To detect any possible RT-PCR inhibition, a spike-in internal control (LightMix Modular EAV RNA Extraction Control, Roche, Basel) was added to the extracted RNA samples. The 70 bp long fragment from the Equine Arteritis Virus genome was amplified with specific primers included in the kit and detected by an Atto647 labelled hydrolysis probe. Limit of detection of the RT-PCR was measured using log10 serial dilution of the EURM-19 copy control.