medRxiv preprint doi: https://doi.org/10.1101/2020.07.08.20147561; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP Steffen Klein1,2,#, Thorsten G. Müller1,#, Dina Khalid1, Vera Sonntag-Buck1, Anke-Mareil Heuser1, Bärbel Glass1, Matthias Meurer3,4, Ivonne Morales5, Angelika Schillak1, Andrew Freistaedter1, Ina Ambiel1, Sophie L. Winter1,2, Liv Zimmermann1, Tamara Naumoska1, Felix Bubeck1, Daniel Kirrmaier3,4, Stephanie Ullrich1, Isabel Barreto Miranda1, Simon Anders3, Dirk Grimm1,6, Paul Schnitzler1, Michael Knop3,4,7, Hans-Georg Kräusslich1,6, Viet Loan Dao Thi1,2, Kathleen Börner1,6,* and Petr Chlanda1,2,* 1) Center of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany 2) Schaller Research Groups, Center of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany 3) Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany 4) German Cancer Research Center (DKFZ), Heidelberg, Germany 5) Center of Infectious Diseases, Clinical Tropical Medicine, Heidelberg University Hospital, Heidelberg, Germany 6) German Center for Infection Research (DZIF), Heidelberg, Germany 7) DKFZ-ZMBH Alliance, Heidelberg, Germany # Equal contribution. * Correspondence: [email protected]; [email protected] Abstract 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 alternative, 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 RT-LAMP using N primers, as well as RT-qPCR using E gene primers showing that the here presented RNA extraction protocol 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. NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.07.08.20147561; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1. Introduction SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19) was first described in the city of Wuhan in China in December 2019 and spread globally thereafter to cause a pandemic. To slow its spread, large-scale diagnostics and the enforcement of strict public health measures were implemented in many countries. The current standard test for SARS‐CoV-2 detection and diagnosis is based on viral RNA extraction from a pharyngeal swab followed by the highly sensitive reverse transcription and quantitative PCR (RT-qPCR) method. Several primer sets targeting one or more of the SARS-CoV-2 genes – N, E, S or RNA-dependent RNA polymerase (RdRp) – have been used [1]. A two-step testing procedure using primer sets targeting the E gene for initial screening followed by the RdRp gene to confirm positive samples is recommended by the German Consiliary Laboratory for Coronaviruses [2]. The unprecedented global demand for commercial RNA extraction kits and the ensuing shortage of these reagents [3], led to the establishment of several diagnostic workflows performed on patient samples with or without an intermediate RNA extraction step [4–7]. Viral RNA isolation from clinical samples depends on the rapid inactivation of viral particles, typically by detergent solubilization and on the denaturation of omnipresent RNases [8]. The latter may be accomplished by the use of chaotropic chemicals, such as guanidinium salts [9] or non-specific proteases that are active on both native and denatured proteins, such as proteinase K. In either case, after virus particle lysis, RNA must be purified since guanidinium salts, proteinase K and organic solvents inhibit the subsequent RT- qPCR step. RNA can be separated from proteins either by liquid phase separation using chloroform-aqueous emulsions after lysis with a commercially available Trizol (a mixture of guanidinium thiocyanate and acid phenol) or by means of solid-phase separation using silica [10]. Nucleic acid-binding to negatively charged silica (SiO2) is facilitated by guanidinium salts and the basic pH of the lysis buffer [11]. To achieve a high nucleic acid binding capacity, silica-based nucleic acid extraction methods use either porous silica matrices embedded in a column (spin column) [12], a tip (TruTips) [13] or a suspension of microparticles. Microparticles can be separated from the lysate either by centrifugation or by a magnetic field if the microparticles' dense iron-containing cores are coated with porous silica [14]. The protocol established in this study aimed at extracting SARS-CoV-2 RNA from respiratory patients' swabs (oropharyngeal and nasopharyngeal) and is based on the magnetic bead- based nucleic acid extraction protocol published by He et al. [15] and on the protocol from Chemicell GmbH, who provided the SiMAG-N-DNA magnetic beads. We opted for silica magnetic beads because of their relatively easy manufacturing and sustainable availability, and because specialized plasticware (spin columns, modified tips) is not required to perform their separation. Magnetic bead RNA extraction was performed in 96-well plates using a magnet plate optimized for 96 deep-well plates. To minimize the pipetting and handling errors, the portable manual pipetting system Liquidator 96, which is less expensive than automated pipetting robots, was used (Figure 1A). Here, we show that the magnetic bead-based protocol yields RNA extracts comparable to the commercially available QIAcube viral RNA extraction kit, as determined by the 2 medRxiv preprint doi: https://doi.org/10.1101/2020.07.08.20147561; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. commonly applied detection methods RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP) [16]. 2. Materials and Methods 2.1 Clinical samples and sample lysis A selection of upper respiratory tract specimens (flocked swabs in Amies medium, eSwab Copan, Brescia, Italy) sent to the diagnostics laboratory of the Heidelberg University Hospital between April and May 2020 for SARS-CoV-2 PCR were used for the study. 17 out of 77 tested positive and 60 tested negative for SARS-CoV-2 by RT-qPCR. In this study, we used samples from the same day (2 positive, 20 negative) or samples collected on previous days that were stored at -20 °C (15 positive, 40 negative). In order to facilitate replicate testing, positive samples were further diluted and used in replica to generate 88 positive and 76 negative samples. The following steps were performed in a biosafety level 2 (BSL-2) laboratory according to standard microbiological and diagnostic practices. To extract SARS-CoV-2 RNA from pharyngeal swabs, a lysis buffer containing 5 M guanidinium thiocyanate, 40 mM dithiothreitol, 20 µg/ml glycogen, 1 % Triton X-100 and buffered with 25 mM sodium citrate to pH 8 was used. Internal control (IC) for RT-qPCR (5 µl/sample) (Tib-Molbiol, Germany) was added into the lysis buffer just before use. To ensure both rapid virus deactivation and RNase denaturation, 140 µl lysis buffer and 140 µl sample were vigorously vortexed in a 1.5 ml Eppendorf tube for 10 sec and incubated for 10 min at room temperature inside a BSL-2 laminar flow cabinet. Lysates (280 µl) were transferred into a 96 deep-well plate (Greiner AG, Austria) with a maximum working volume of 500 µl per well, which is compatible with the Liquidator 96 pipetting system. 2.2 RNA extraction using magnetic beads in 96-well plate format Just before the RNA extraction, SiMAG-N-DNA magnetic beads (Chemicell, Germany) were washed three times in RNase-free water. The aqueous magnetic bead stock solution of 100 µg/µl was added into absolute ethanol to obtain a working solution of 5 µg/µl. Using a multichannel pipette, 200 µl of magnetic bead solution was transferred into the 96 deep-well plate containing the pharyngeal sample lysates. To facilitate the adsorption of the nucleic acid onto the magnetic beads, the 96-deep-well plate was placed on an orbital shaker MS3 (IKA, Germany) for 8 min at 500 – 1000 rpm, before the mixture was resuspended using a Liquidator 96, Model 200 µl (Mettler Toledo, USA) (Figure 1A,B) and placed again on the orbital shaker for additional 7 min. Subsequently the plate was placed on a magnet plate for 96-deep-well plates (Magtivio, Netherlands) (Figure 1C) for 10 min to allow the magnetic beads to form rings on the bottom of the wells (Figure 1D,E). The clear supernatant was discarded using the Liquidator 96 and magnetic beads were washed three times with 200 µl 70 % ethanol and briefly rinsed with 50 µl RNase-free water to remove any residual ethanol.