The Altona Diagnostics
RealStar SARS-CoV-2 RT-PCR Kit 1.0 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 30 μL (10 μL extracted sample and 20 μL Master Mix). The kit contains 2 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
Supplemental Table 2. Taqman RT-PCR was performed using the
Prism 7500 Sequence Detection System (Applied Biosystems) 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 and then 72.0°C for 15 seconds. Validation of this assay was performed as described in ref. 22 (
link).
Testing on the Cepheid
Xpert Xpress SARS-CoV-2 GeneXpert platform was performed in accordance with manufacturer’s instructions (40 ).
Thielen P.M., Wohl S., Mehoke T., Ramakrishnan S., Kirsche M., Falade-Nwulia O., Trovão N.S., Ernlund A., Howser C., Sadowski N., Morris C.P., Hopkins M., Schwartz M., Fan Y., Gniazdowski V., Lessler J., Sauer L., Schatz M.C., Evans J.D., Ray S.C., Timp W, & Mostafa H.H. (2021). Genomic diversity of SARS-CoV-2 during early introduction into the Baltimore–Washington metropolitan area. JCI Insight, 6(6), e144350.