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

Steffen Klein,Matthias Meurer,Petr Chlanda,Liv Zimmermann,Andrew Freistaedter,Michael Knop,Daniel Kirrmaier,Dirk Grimm,Ina Ambiel,Paul Schnitzler,Bärbel Glass,Dina Khalid,Isabel Barreto Miranda,Simon Anders,Ivonne Morales,Felix Bubeck,Thorsten G Müller,Viet Loan Dao Thi,Tamara Naumoska,Kathleen Börner,Stephanie Ullrich,Angelika Schillak,Sophie L Winter,Hans‐Georg Kräusslich ,Vera Sonntag‐Buck ,Anke‐Mareil Heuser

doi:10.3390/v12080863

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, 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.

Highlights

Severe acute respiratory syndrome coronavirus 2 (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 causing pandemic
The magnetic bead RNA extraction protocol was established in a 96-well plate format as part of the detection workflow (Figure 2)
Processing using a magnetic bead purification protocol for SARS-CoV-2 diagnostics. We first applied both protocols on one SARS-CoV-2 positive pharyngeal swab sample, which was diluted in RNase-free water prior to RNA isolation in a 10-fold dilution series up to 105 fold, in

Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (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 causing pandemic. The current standard test for SARS-CoV-2 detection and diagnosis is based on viral RNA extraction from a pharyngeal swab followed by highly sensitive reverse transcription and quantitative PCR (RT-qPCR). Several primer sets targeting one or more of the SARS-CoV-2 genes—nucleocapsid (N), envelope protein (E), S glycoprotein (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 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 [4,5,6,7]. Viral RNA isolation from clinical samples depends on the rapid inactivation of viral particles, typically by detergent solubilization, and on the denaturation of omnipresent

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