Hydrolysis probe

An in-house SARS-CoV-2 RT-PCR protocol and a commercial RT-PCR kit were used for method comparison. Since both assays work only with purified RNA, the above-mentioned RNA isolation procedure was mandatory prior to PCR testing.
The in-house PCR protocol was based on the QuantiTect Probe RT-PCR Kit (Qiagen, Hilden, Germany) with primers and a hydrolysis probe (Biomers, Ulm, Germany) targeting the E gene. Detection was done on the FAM channel of a LightCycler 96 instrument (Roche, Basel, Switzerland). Primer sequences and the temperature profile are shown in the appendix (Supplementary Information 1).
For comparison of the SARS-CoV-2 RT-LAMP protocol with a commercial kit, the LABSYSTEMS COVID-19 Real Time Multiplex RT-PCR Kit (Labsystems Diagnostics OY, Helsinki, Finland) was used. This kit is designed to detect three genes (ORF1ab, E, N) of the SARS-CoV-2 genome simultaneously. The RT-PCR reactions were set up according to the manufacturer’s protocol and were analysed on a Rotor-Gene Q instrument.

Viral RNA was isolated from nasopharyngeal swaps using PrepitoViral DNA/RNA300 isolation kits (PerkinElmer, Waltham, USA). PCR testing was performed by using the QuantiTect Probe RT-PCR Kit (Qiagen, Hilden, Germany) with primers and a hydrolysis probe (Biomers, Ulm, Germany) targeting the E gene (Suppl. Material 1). Detection was done on the FAM channel of a LightCycler 96 instrument (Roche, Basel, Switzerland).