The following report describes three different systems for the detection of SARS-CoV-2 using reverse transcription and subsequent amplification of virus-specific nucleic acid.
The system currently used by AGES detecs the viral E and RdRp genes with internal control (EAV, Equine Arteritis Virus) according to the WHO recommendation: Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR (Victor M Corman et al., 2020) and is a “homebrew” system in which primers, probes and controls from TIB-MolBiol (Berlin) and buffer/enzyme from Invitrogen SuperScriptTM III One-Step RT-PCR System (ThermoFischer) are used.
In the first approach (“screening”), a 76 base pair fragment of the viral E gene and the internal control (EAV, extraction and PCR control) are detected. Positive samples in E-gene PCR are additionally tested with RdRp (100 base pairs) for confirmation. The second system, a “rapid test” with isothermal amplification (MIRAI GENOMICS), uses the SmartAmp 2nd Kit from RIKEN (Japan). The third test kit -ViroReal® Kit SARS-CoV-2 & SARS (Ingenetix, Vienna) uses the viral N gene as the target sequence.
The three systems tested matched 90 of the 94 patient samples. In two cases (samples 850500 and 850514) the confirmation test of positive E gene with RdRp was negative (routine system AGES), but both MIRAI and Ingenetix were positive. The “Cp values” for the isothermal amplification were very high (75.25 and 82.32). Both MIRAI (850461) and Ingenetix (850502) gave a positive result with negative E-and RdRp genes.
Florian Gruber, MedUni Vienna
Peter Hufnagl, AGES
link: ages.at