Viral RNA extraction was performed from 300 μL viral transport medium using the column-based Quick-RNA viral kit (Zymo Research, Orange, CA) following the manufacturer’s instructions. SARS-CoV-2 viral RNA was detected using the Luna Universal Probe one-step reverse transcription-quantitative PCR (RT-qPCR) kit (New England Biolabs, MA, USA). The reaction mixture included 7 μL of viral RNA, the oligonucleotides and probe for the E gene, and the conditions reported in the Berlin real-time RT-PCR protocol v2 (15 (link)) with a thermal modification in reverse transcription (55°C for 18 min) and in the alignment/extension step (60°C for 30 s), according to the one-step RT-qPCR kit manufacturer’s recommendations.
In addition, human RNase P gene transcripts were detected as an internal control and for evaluation of the quality of the sample, as previously recommended (37 (link)). The RT-PCRs were carried out in a CFX-96 Bio-Rad thermal cycler (Bio-Rad, CA, USA). Tests were performed in parallel with a negative control (sample replaced by water) and a positive control (RNA from virus isolated at the University of Antioquia) (38 (link)).
In addition, human RNase P gene transcripts were detected as an internal control and for evaluation of the quality of the sample, as previously recommended (37 (link)). The RT-PCRs were carried out in a CFX-96 Bio-Rad thermal cycler (Bio-Rad, CA, USA). Tests were performed in parallel with a negative control (sample replaced by water) and a positive control (RNA from virus isolated at the University of Antioquia) (38 (link)).