Relative quantification of SARS-CoV-2 viral load using RT-qPCR
Saliva samples were thawed at room temperature and prior to nucleic acid extraction, saliva samples were spiked with Internal Extraction Control RNA (IEC; an exogenous RNA of rat Phogrin gene, NM_031600, amplicon 98 bp) from the genesig COVID-19 qPCR Assay kit (PrimerDesign Ltd., UK) to verify the successful extraction in case of a negative SARS-CoV-2 result (Fig. One-step RT-qPCR workflow for quantification of salivary SARS-CoV-2 viral load using MagMAX Viral/Pathogen Nucleic Acid Isolation Kit, genesig COVID-19 qPCR Assay. Figure created using CorelDRAW Graphics Suite 2017 (version 19.1.0.419) SARS-CoV-2 viral RNA were purified from saliva samples (100–200 µL) using MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific Inc; Cat. A42352) according to manufacturer’s protocol. SARS-CoV-2 viral load were measured using a one-step RT-qPCR kit (genesig COVID-19 qPCR Assay, PrimerDesign, Cat. Z-Path-2019-nCov). As per genesig COVID-19 qPCR assay protocol, SARS-CoV-2 RdRp gene (C19, with FAM 465-510 probe), rat Phogrin gene (IEC, with VIC/HEX 533-580 probe) and a human endogenous control (HEC, with FAM 465-510 probe; ATCB gene, NM_001101, amplicon 92 bp) were measured from each eluted saliva RNA sample in quadruplicate RT-qPCR reactions using a LightCycler LC480 Instrument (Roche Diagnostics, UK). RT-qPCR amplification began with reverse transcription at 55 °C (10 min) followed by Hot-start activation at 95 °C (2 min) prior to 45 cycles of 95 °C (10 s) denaturation, 60 °C (60 s) annealing/extension/acquisition. Every qPCR assay plate included quadruplicate wells of no saliva extraction control, no template control and positive template control for assay quality assurance. The IEC exogenous RNA was used as a positive control for the nucleic acid extraction process. Successful IEC co-detection with SARS-CoV-2 would indicate that PCR inhibitors were not present at a high concentration. Samples with poor IEC amplification (crossing point; Cp > 45) were disqualified as these represented poor RNA extraction (containing inhibitors that interfered with qPCR). HEC gene was measured to confirm successful extraction of a valid biological sample of human origin. Samples with poor HEC amplification (Cp > 45) were disqualified as these indicated samples of insufficient human biological RNA yield. Viral load was calculated from sample Cp/Cq ratio values against a standard curve of Log viral load vs crossing point (Fig. SARS-CoV-2 viral load were normalised against both IEC and HEC to control for RNA quality and human RNA loading, respectively, in each sample. Samples with undetectable viral levels were assigned a Cp value of 45 for calculation.