Determination of normalized viral load blinded to treatment arm was performed on nasopharyngeal swab and lower respiratory tract specimens by RNA extraction on the EMAG® platform (bioMerieux, Marcy-l'Étoile, France). The SARS-CoV-2 load was measured by quantitative RT-PCR, according to a scale of calibrated in-house plasmid, using the RT-PCR RdRp-IP4 developed by the Institut Pasteur (Paris, France) [16 ]. The amplification protocol was developed using QuantStudio 5 rtPCR Systems (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The number of cells in a sample (quality criteria for nasopharyngeal swab and normalization tool for viral load determination) was checked using the CELL Control r-gene® kit (Argene-BioMérieux, Marcy-l'Étoile, France). If cell quantification was <500 cells/reaction, the quality of the sample was considered too low to be measured. We computed a normalized SARS-CoV-2 load by dividing the viral load by the number of cells. All viral loads strictly below 1 log10 RNA copies/10 000 cells were considered under the limit of detection and were reported as negative.
Quantstudio 5 rtpcr systems
Manufactured by Thermo Fisher Scientific
Sourced in United States
The QuantStudio 5 rtPCR Systems is a real-time PCR instrument designed for quantitative gene expression analysis. It provides accurate and precise measurement of target DNA sequences in samples. The system features a 96-well format and is compatible with a variety of chemistry options and sample types.
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3 protocols using quantstudio 5 rtpcr systems
1
SARS-CoV-2 Viral Load Quantification
2
Quantitative SARS-CoV-2 RT-PCR Protocol
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Determination of normalized viral load was performed on plasma samples by RNA extraction on the EMAG® platform (bioMerieux, Marcy-l'Étoile, France). The SARS-CoV-2 load was measured by quantitative RT-PCR, according to a scale of calibrated in-house plasmid, using the RT-PCR RdRp-IP4 developed by the Institut Pasteur (Paris, France).7 The amplification protocol was developed using QuantStudio 5 rtPCR Systems (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The absence of inhibitors in the specimen was checked by using the RNA Internal Control R-GENE® kit (Argene_BioMérieux, Marcy-l'Étoile, France) on each sample. We expressed normalized SARS-CoV-2 load in log10 RNA copies/mL and all viral loads strictly below 4 RNA copies/reaction were considered under the limit of detection and were reported as negative.
3
BALF RNA Extraction and Quantification
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RNA extraction was performed on a NucliSENS® easyMAG® platform (Biomerieux, Marcy-l’Étoile, France), from 200 μL of cell-free BALF, and according to the manufacturer’s instructions. Two target genes were amplified and tested simultaneously, namely the RNA-dependent RNA polymerase (RDRP) IP2 and IP4 regions [16 ]. Amplification was performed using QuantStudio 5 rtPCR Systems (Thermo Fisher Scientific, Waltham, MA, USA). Quantification of the number of RNA copies was done according to a scale ranging from 103 to 106 copies/mL. All positive BALF samples were quantified and expressed as the number of RNA copies/μL.
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