tetano
Editor, Senior Moderator
Viruses
. 2020 Aug 7;12(8):E863.
doi: 10.3390/v12080863.
SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP
Steffen Klein[SUP] 1 2 [/SUP], Thorsten G M?ller[SUP] 1 [/SUP], Dina Khalid[SUP] 1 [/SUP], Vera Sonntag-Buck[SUP] 1 [/SUP], Anke-Mareil Heuser[SUP] 1 [/SUP], B?rbel Glass[SUP] 1 [/SUP], Matthias Meurer[SUP] 3 4 [/SUP], Ivonne Morales[SUP] 5 [/SUP], Angelika Schillak[SUP] 1 [/SUP], Andrew Freistaedter[SUP] 1 [/SUP], Ina Ambiel[SUP] 1 [/SUP], Sophie L Winter[SUP] 1 2 [/SUP], Liv Zimmermann[SUP] 1 [/SUP], Tamara Naumoska[SUP] 1 [/SUP], Felix Bubeck[SUP] 1 [/SUP], Daniel Kirrmaier[SUP] 3 4 [/SUP], Stephanie Ullrich[SUP] 1 [/SUP], Isabel Barreto Miranda[SUP] 1 [/SUP], Simon Anders[SUP] 3 [/SUP], Dirk Grimm[SUP] 1 6 [/SUP], Paul Schnitzler[SUP] 1 [/SUP], Michael Knop[SUP] 3 4 7 [/SUP], Hans-Georg Kr?usslich[SUP] 1 6 [/SUP], Viet Loan Dao Thi[SUP] 1 2 [/SUP], Kathleen B?rner[SUP] 1 6 [/SUP], Petr Chlanda[SUP] 1 2 [/SUP]
Affiliations
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.
Keywords: COVID-19; RNA virus; RT-LAMP; RT-qPCR; SARS-CoV-2; coronavirus; diagnostics; high-throughput screening; magnetic bead RNA purification; pandemic.
. 2020 Aug 7;12(8):E863.
doi: 10.3390/v12080863.
SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP
Steffen Klein[SUP] 1 2 [/SUP], Thorsten G M?ller[SUP] 1 [/SUP], Dina Khalid[SUP] 1 [/SUP], Vera Sonntag-Buck[SUP] 1 [/SUP], Anke-Mareil Heuser[SUP] 1 [/SUP], B?rbel Glass[SUP] 1 [/SUP], Matthias Meurer[SUP] 3 4 [/SUP], Ivonne Morales[SUP] 5 [/SUP], Angelika Schillak[SUP] 1 [/SUP], Andrew Freistaedter[SUP] 1 [/SUP], Ina Ambiel[SUP] 1 [/SUP], Sophie L Winter[SUP] 1 2 [/SUP], Liv Zimmermann[SUP] 1 [/SUP], Tamara Naumoska[SUP] 1 [/SUP], Felix Bubeck[SUP] 1 [/SUP], Daniel Kirrmaier[SUP] 3 4 [/SUP], Stephanie Ullrich[SUP] 1 [/SUP], Isabel Barreto Miranda[SUP] 1 [/SUP], Simon Anders[SUP] 3 [/SUP], Dirk Grimm[SUP] 1 6 [/SUP], Paul Schnitzler[SUP] 1 [/SUP], Michael Knop[SUP] 3 4 7 [/SUP], Hans-Georg Kr?usslich[SUP] 1 6 [/SUP], Viet Loan Dao Thi[SUP] 1 2 [/SUP], Kathleen B?rner[SUP] 1 6 [/SUP], Petr Chlanda[SUP] 1 2 [/SUP]
Affiliations
- PMID: 32784757
- 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.
Keywords: COVID-19; RNA virus; RT-LAMP; RT-qPCR; SARS-CoV-2; coronavirus; diagnostics; high-throughput screening; magnetic bead RNA purification; pandemic.