SARS-CoV-2 RT-qPCR Quantification Protocol

Viral RNA was automatically extracted from 200 μL of the NPS specimens using the MagMAX^TM Viral/Pathogen II Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Vilnius, Lithuania) on KingFisher (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RT-qPCR was performed using TaqPath™ COVID-19 multiplex real-time RT-PCR test (the Orf1ab, N and S genes) (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Negative and positive controls were run simultaneously with samples [2 (link)]. The assay was performed using a QuantStudio 5 real-time PCR detection system (Thermo Fisher Scientific, Waltham, MA, USA). Test results were reported quantitatively as cycle threshold (Ct) value of the ORF1ab, the N and the S genes, while qualitative data were reported as positive/negative at test cut-off (i.e., Ct < 37 for positive or ≥ 37 for negative samples). The Ct values used to classify specimens as ‘high viral load’, ‘medium viral load’ and ‘low viral load’ defined as previously described [7 (link),21 (link)], they were <18.57, 18.57–28.67 and >28.67, respectively.