Rnadvance viral reagent kit

Naso/oropharyngeal swab samples (FLOQSwab; Copan, Brescia, Italy) for detection of SARS-CoV-2 RNA were collected in 3 mL sterile universal transport medium (UTM; Copan) and stored at 4 °C for up to four days prior to testing. Viral RNA was extracted from 200 µL sample using the RNAdvance Viral Reagent Kit (Beckman Coulter Inc., Brea, CA, USA) in a Biomek i7 Automated Workstation (Beckman Coulter Inc.), according to the manufacturer’s instructions. Real-time reverse transcriptase PCR was performed in a QuantStudio 6 system (Applied Biosystems, Beijing, China) targeting the envelope (E) gene specific to the Sarbecovirus [17 (link)]. The limit of detection was 20 viral RNA genome copies/mL. The results were reported as positive (Ct values ≤ 40) or negative (Ct values > 40).

For small-scale experiments, viral genomes from throat swabs collected in 1xPBS (700 μL) were isolated using MagNa Pure 96 nucleic acid extraction system (Roche) with reagents from the MagNa Pure 96 DNA and Viral NA Small Volume kit with 200 μL sample in 1xPBS as input and 100 μL elution. As positive control material, supernatant from SARS-CoV-2 infected cells (120 μL) were mixed with 120 μL of MagNA Pure Lysis Buffer (Roche) followed by extraction as small-scale SARS-CoV-2 patient samples. Positive control RNA was stored at -80°C until use. For large-scale SARS-CoV-2 patient screening (Variant-PCR), viral RNA was extracted using Beckman Coulter RNAdvance Viral Reagent kit with 200 μl sample in 1xPBS as input and 50 μl Nuclease-free water for elution on Beckman Coulter Biomek i7 automated workstations.