Realstar sars cov 2 rt pcr kit 1

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 .

SARS-CoV-2 Ag-RDT (index test): Antigen testing was conducted from anterior nasal swabs by the Veritor™ System for Rapid Detection of SARS-CoV-2 (Becton, Dickinson and Company, Franklin Lake, NJ, USA). This test has a limit of detection of 140 50% tissue culture infective doses of nucleocapsid protein and was used according to the manufacturer's instructions (18 ). Briefly, swabs were added to extraction reagent tubes at room temperature within 60 min after sampling and mixed for at least 15s. Three drops extraction buffer/specimen mixture were then added to a test cartridge. After 15 min of incubation, the test devices were interpreted using a Veritor™ Analyzer.
RT-PCR (reference standard): RNA was extracted from NPS and purified using GenePure Pro, Nucleic Acid Purification System (Bioer Technology, Hangzhou, China). qRT-PCR was performed using the RealStar^® SARS-CoV-2 RT-PCR kit 1.0 (Altona Diagnostics, Hamburg, Germany) on a Roche Cobas z 480 RT-PCR device.

All self-collected specimens were tested at the Institute of Medical Virology, Goethe University Frankfurt. Dry swabs (nasal swab, tongue swab) were suspended in 2 mL of phosphate-buffered saline (PBS) and incubated for 5 min prior to further processing. Depending on the viscosity in the self-collected specimen and to achieve the required input volume, saliva was diluted up to 1:2.5. The chewed cotton pads with saliva were suspended in 4 mL of PBS, compressed, and incubated for 5 min. Gargle solution was used native, without further dilution. Samples were stored at −80 °C until nucleic acid extraction and PCR-based testing was performed. Specimens were extracted using the QIAsymphony and the DSP virus/pathogen midi kit (both Qiagen GmbH, Hilden, Germany) according to the manufacturers’ protocols. Realtime RT-PCR (rRT-PCR) analysis was performed using the RealStar^® SARS-CoV-2 RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany) [17 (link)] on the ABI PRISM^® 7500 Analyser (Applied Biosystems, Waltham, MA, USA) according to the manufacturers´ specifications. Three quantitative comparison samples containing 10⁵, 10⁶, and 10⁷ SARS-CoV-2 (BetaCoV/Munich/ChVir984/2020) RNA copies/mL were used to generate a standard curve and to calculate the viral RNA copies/mL. Equivocal test results were excluded from the analysis of testing sensitivity.

During the study period, the Henri Mondor hospital virology laboratory used several platforms for SARS-CoV-2 RNA testing, based on technical developments and reagent availability. Between March and June 2020, samples were tested using an in-house RT-qPCR assay based on the Charité protocol targeting the E and RNA-dependent RNA polymerase (RdRp) genes (16 (link)) and on a commercially available RT-PCR assay targeting the E and S genes (RealStar SARS-CoV-2 RT PCR kit 1.0; Altona Diagnostics GmbH, Hamburg, Germany) (17 (link)). After September 2020, SARS-CoV-2 RNA was detected by means of commercially available molecular biology technologies, including TMA (Aptima SARS CoV-2 on the Panther system; Hologic, Marlborough, MA) or RT-PCR (Alinity m; Abbott Molecular, Des Plaines, IL). Samples found to be RNA positive in TMA were retested by RT-PCR (TaqPath COVID-19 RT-PCR kit; Thermo Fisher, Pleasanton, CA) to measure C_T values. Samples with C_T values above 40 were considered negative.