Molecular Diagnostics of SARS-CoV-2 Using RT-PCR

The Altona Diagnostics RealStar® SARS-CoV-2 RT-PCR Kit 1.0 (Hamburg, Germany) was the primary assay used for molecular diagnosis. A subset of SARS-CoV-2 positives were identified using the Cepheid Xpert Xpress SARS-CoV-2 GeneXpert platform per manufacturer instructions. All samples were processed within 24 hours of collection.
The RealStar® SARS-CoV-2 RT-PCR Kit 1.0 total reaction volume was 30μL (10μL extracted sample and 20μL Master Mix). The kit contains two premade master mixes, A and B, which contain PCR buffer, magnesium salt, primers and probes, reverse transcriptase, and DNA polymerase. The detectors used are Cy5 (SARS-CoV-2; S gene), FAM (B-βCoV; E gene) and JOE (Internal Control). C_T values for the S gene target (Cy5) are reported in Table S2. Taqman RT- PCR was performed using the Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) at the following cycling conditions: 1 cycle at 55.0°C for 20 minutes, 1 cycle at 95.0 °C for 2 minutes and 45 cycles at 95.0°C for 15 seconds, 55.0°C for 45 seconds then 72.0 °C for 15 seconds. Validation of this assay was performed as described in^{22 (link)}.
Testing on the Cepheid Xpert Xpress SARS-CoV-2 GeneXpert platform was performed in accordance with manufacturers instructions³⁴ .