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

• Published in: Viruses Vol. 12; no. 8; p. 863
• Main Authors: Klein, Steffen, Müller, Thorsten G, Khalid, Dina, Sonntag-Buck, Vera, Heuser, Anke-Mareil, Glass, Bärbel, Meurer, Matthias, Morales, Ivonne, Schillak, Angelika, Freistaedter, Andrew, Ambiel, Ina, Winter, Sophie L, Zimmermann, Liv, Naumoska, Tamara, Bubeck, Felix, Kirrmaier, Daniel, Ullrich, Stephanie, Barreto Miranda, Isabel, Anders, Simon, Grimm, Dirk, Schnitzler, Paul, Knop, Michael, Kräusslich, Hans-Georg, Dao Thi, Viet Loan, Börner, Kathleen, Chlanda, Petr
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 07.08.2020; MDPI
• Subjects: Acids; Betacoronavirus - chemistry; Betacoronavirus - genetics; Betacoronavirus - isolation & purification; Clinical Laboratory Techniques - methods; Colorimetry; coronavirus; Coronavirus Infections - diagnosis; Coronavirus Infections - virology; Coronaviruses; COVID-19; COVID-19 Testing; diagnostics; E protein; Ethanol; Gene amplification; Humans; Laboratories; Magnetic Phenomena; Molecular Diagnostic Techniques - methods; N gene; Nucleic Acid Amplification Techniques - methods; Pandemics; Pharynx; Pneumonia, Viral - diagnosis; Pneumonia, Viral - virology; Real-Time Polymerase Chain Reaction - methods; Reverse Transcription; Ribonucleic acid; RNA; RNA polymerase; RNA, Viral - genetics; RNA, Viral - isolation & purification; RT-LAMP; RT-qPCR; SARS-CoV-2; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Switzerland; Germany; COVID-19; RT-LAMP; SARS-CoV-2; diagnostics; RNA virus; high-throughput screening; magnetic bead RNA purification; coronavirus; pandemic; RT-qPCR
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v12080863

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.