RT-qPCR Assay for SARS-CoV-2 Detection in Swabs and Saliva

RT-qPCR amplification assays for swabs and saliva were carried out using reagents from AgPath-ID™ One-Step RT-PCR (Thermo Fisher Scientific, Waltham, Massachusetts, USA). A set of probe-associated primers (assay) aimed at SARS-CoV-2 nucleocapsid (N1 and N2) and envelope (E) genes was used [18 ,19 (link)]. Endogenous control Ribonuclease P/MRP Subunit P30 (RPP30) was also included into the assay to assess sample integrity. The RT-qPCR mixture contained 10 µM of sense and antisense primers, 5 µM of probe, 2X RT-PCR buffer, 25X RT-PCR Enzyme mix (ArrayScript™ Reverse Transcriptase and AmpliTaq Gold^® polymerase), and 5 µL of viral RNA extracted from swabs and 7 µL from saliva. The conditions for the RT-qPCR at Rotor-Gene Q equipment (QIAGEN) were as follows: 45 °C for 15 min for reverse transcription followed by enzyme activation at 95 °C for 10 min, and then 45 cycles were conducted at 95 °C for 15 s and at 55 °C for 45 s. Ct values below 40 for two of the three genes represented positive results. Fluorescence readings were performed using the FAM channel, and the analysis of the results was performed using the Rotor-gene Q series program with the threshold set at 0.1.