Ncovpc

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 ).

For Ribonucleic acid (RNA) extraction, the commercial QIAamp^® Viral RNA Mini Kit (Qiagen, Germany) was used, following the manufacturer’s protocol. Samples were submitted to real-time polymerase chain reaction post reverse transcription (RT-qPCR) after the RNA extraction, using the Promega GoTaq^® Probe 1-Step RT-qPCR System, according to the manufacturer’s protocol [33 (link)]. The primers and probes targeted two SARS-CoV-2 regions of the N gene (N1 and N2), the human RNAse P (RP) gene, and IDT (Integrated DNA Technologies, Coralville, IA, USA). All samples that presented a cycle threshold (Ct) lower than 40 (for N1, N2, and RP targets) were considered positive for SARS-CoV-2 RNA. 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: 189 NC_045512.2) from Integrated DNA Technologies [34 ] was used to estimate the viral loads in genomic copies (GCs) per mL/g of clinical specimens.