Gotaq probe 1 step rt qpcr system

RNA isolation from lung and nasal turbinate samples, RNA quantification, and DNAse treatment has been detailed previously.²⁵ The viral RNA derived from the lung and nasal turbinate samples was quantified using a protocol for quantifying the SARS-CoV-2 sub-genomic E gene RNA (sgE)^{29 (link)} using the GoTaq^® Probe 1-Step RT-qPCR System (Promega).
Quantification of SARS-CoV-2 E SgRNA was completed utilising primers and probes previously described elsewhere^{29 (link)} and were used at 400 nM and 200 nM, respectively (IDT), using the GoTaq^® Probe 1-Step RT-qPCR System (Promega). Quantification of 18S RNA utilised previously described primers and probe sequences,^{30 (link)} and were used at 300 nM and 200 nM, respectively (IDT), using the GoTaq^® Probe 1-Step RT-qPCR System (Promega). Methods for the generation of the 18S and sgE RNA standards have been outlined previously.^{31 (link)} Both PCR products were serially diluted to produce standard curves in the range of 5 × 10⁸ − 5 copies/reaction via a 10-fold serial dilution. DNAse treated RNA at 20,000 ng/mL or dH₂O were added to appropriate wells producing final reaction volumes of 20 μL. The prepared plates were run using a qTOWER³ Real-Time PCR Detector (Analytik Jena). Thermal cycling conditions have been detailed previously.²⁵ The sgE data were normalised to 18S data for subsequent quantitation.

Ribonucleic acid (RNA) extraction was performed using the commercial QIAamp® Viral RNA Mini Kit (Qiagen, Germany), following the manufacturer’s protocol. After RNA extraction, samples were submitted to real-time polymerase chain reaction post reverse transcription (RT-qPCR) using the Promega GoTaq® Probe 1-Step RT-qPCR System, according to the manufacturer’s protocol (23 (link)). Primers and probes targeted two regions of the N gene (N1 and N2) from SARS-CoV-2 and the human RNAse P (RP) gene, and IDT (Integrated DNA Technologies, Iowa, USA). All samples that presented a cycle threshold (Ct) lower than 40 (for N1, N2, and RP targets) were positive for SARS-CoV-2 RNA. Viral loads in genomic copies (GC) per mL/g of clinical specimens were estimated based on a standard curve of serial dilutions (10⁶ to 10⁰ GC/µL) of the synthetic positive control nCoVPC (severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, GenBank: NC_045512.2) from Integrated DNA Technologies (24 ).

Materials were purchased and used as received without further purification: chloroform, isopropanol, ethanol, phosphate buffered saline (PBS) and nuclease-free water were purchased from Fisher Scientific (UK). Male K18-hACE2 mice were purchased from Charles River (France). Ronapreve (casirivimab and imdevimab) was kindly provided by Roche (Switzerland). TRIzol^™, GlycoBlue^™, Phasemaker^™ tubes and TURBO DNA-free^™ kit were purchased from Fisher Scientific (UK). GoTaq^® Probe 1-Step RT-qPCR System was purchased from Promega (US). SARS-CoV-2 (2019nCoV) CDC qPCR Probe Assay was purchased from IDT (US). Precellys CKmix lysing tubes were purchased from Bertin Instruments (France). For immunohistology, a rabbit anti-SARS-CoV nucleoprotein antibody was purchased from Rocklands, the peroxidase blocking buffer and the Envision+System HRP Rabbit and the diaminobenzidine from Agilent/DAKO. All other chemicals and reagents were purchased from Merck (UK) and used as received, unless stated otherwise.