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SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP

1
Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany
2
Schaller Research Groups, Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany
3
Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany
4
German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany
5
Center of Infectious Diseases, Clinical Tropical Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany
6
Center of Infectious Diseases, Integrative Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany
7
German Center for Infection Research (DZIF), 69120 Heidelberg, Germany
8
DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Viruses 2020, 12(8), 863; https://doi.org/10.3390/v12080863
Received: 7 July 2020 / Revised: 31 July 2020 / Accepted: 5 August 2020 / Published: 7 August 2020
Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot. View Full-Text
Keywords: coronavirus; SARS-CoV-2; COVID-19; diagnostics; RT-qPCR; RT-LAMP; magnetic bead RNA purification; RNA virus; pandemic; high-throughput screening coronavirus; SARS-CoV-2; COVID-19; diagnostics; RT-qPCR; RT-LAMP; magnetic bead RNA purification; RNA virus; pandemic; high-throughput screening
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Klein, S.; Müller, T.G.; Khalid, D.; Sonntag-Buck, V.; Heuser, A.-M.; Glass, B.; Meurer, M.; Morales, I.; Schillak, A.; Freistaedter, A.; Ambiel, I.; Winter, S.L.; Zimmermann, L.; Naumoska, T.; Bubeck, F.; Kirrmaier, D.; Ullrich, S.; Barreto Miranda, I.; Anders, S.; Grimm, D.; Schnitzler, P.; Knop, M.; Kräusslich, H.-G.; Dao Thi, V.L.; Börner, K.; Chlanda, P. SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP. Viruses 2020, 12, 863.

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