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SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.
Viruses. 2020 08 07; 12(8)V

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.

Authors+Show Affiliations

Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. Schaller Research Groups, Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany. German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.Center of Infectious Diseases, Clinical Tropical Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. Schaller Research Groups, Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany. German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. German Center for Infection Research (DZIF), 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), 69120 Heidelberg, Germany. German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany. DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. German Center for Infection Research (DZIF), 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. Schaller Research Groups, Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. German Center for Infection Research (DZIF), 69120 Heidelberg, Germany.Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany. Schaller Research Groups, Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

32784757

Citation

Klein, Steffen, et al. "SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing By RT-qPCR and RT-LAMP." Viruses, vol. 12, no. 8, 2020.
Klein S, Müller TG, Khalid D, et al. SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP. Viruses. 2020;12(8).
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., ... Chlanda, P. (2020). SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP. Viruses, 12(8). https://doi.org/10.3390/v12080863
Klein S, et al. SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing By RT-qPCR and RT-LAMP. Viruses. 2020 08 7;12(8) PubMed PMID: 32784757.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP. AU - Klein,Steffen, AU - Müller,Thorsten G, AU - Khalid,Dina, AU - Sonntag-Buck,Vera, AU - Heuser,Anke-Mareil, AU - Glass,Bärbel, AU - Meurer,Matthias, AU - Morales,Ivonne, AU - Schillak,Angelika, AU - Freistaedter,Andrew, AU - Ambiel,Ina, AU - Winter,Sophie L, AU - Zimmermann,Liv, AU - Naumoska,Tamara, AU - Bubeck,Felix, AU - Kirrmaier,Daniel, AU - Ullrich,Stephanie, AU - Barreto Miranda,Isabel, AU - Anders,Simon, AU - Grimm,Dirk, AU - Schnitzler,Paul, AU - Knop,Michael, AU - Kräusslich,Hans-Georg, AU - Dao Thi,Viet Loan, AU - Börner,Kathleen, AU - Chlanda,Petr, Y1 - 2020/08/07/ PY - 2020/07/07/received PY - 2020/07/31/revised PY - 2020/08/05/accepted PY - 2020/8/14/entrez PY - 2020/8/14/pubmed PY - 2020/9/10/medline KW - COVID-19 KW - RNA virus KW - RT-LAMP KW - RT-qPCR KW - SARS-CoV-2 KW - coronavirus KW - diagnostics KW - high-throughput screening KW - magnetic bead RNA purification KW - pandemic JF - Viruses JO - Viruses VL - 12 IS - 8 N2 - 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. SN - 1999-4915 UR - https://www.unboundmedicine.com/medline/citation/32784757/SARS_CoV_2_RNA_Extraction_Using_Magnetic_Beads_for_Rapid_Large_Scale_Testing_by_RT_qPCR_and_RT_LAMP_ L2 - https://www.mdpi.com/resolver?pii=v12080863 DB - PRIME DP - Unbound Medicine ER -