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A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.
Sci Transl Med. 2020 08 12; 12(556)ST

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.

Authors+Show Affiliations

Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. vietloan.daothi@med.uni-heidelberg.de m.knop@zmbh.uni-heidelberg.de s.anders@zmbh.uni-heidelberg.de. Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. German Center for Infection Research (DZIF), Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany. German Cancer Research Center (DKFZ), Heidelberg, Germany. DKFZ-ZMBH Alliance, Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany. German Cancer Research Center (DKFZ), Heidelberg, Germany.Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany. DKFZ-ZMBH Alliance, Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. German Cancer Research Center (DKFZ), Heidelberg, Germany.Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany. German Center for Infection Research (DZIF), Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany. vietloan.daothi@med.uni-heidelberg.de m.knop@zmbh.uni-heidelberg.de s.anders@zmbh.uni-heidelberg.de. German Cancer Research Center (DKFZ), Heidelberg, Germany. DKFZ-ZMBH Alliance, Heidelberg, Germany.Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany. vietloan.daothi@med.uni-heidelberg.de m.knop@zmbh.uni-heidelberg.de s.anders@zmbh.uni-heidelberg.de.

Pub Type(s)

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

Language

eng

PubMed ID

32719001

Citation

Dao Thi, Viet Loan, et al. "A Colorimetric RT-LAMP Assay and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples." Science Translational Medicine, vol. 12, no. 556, 2020.
Dao Thi VL, Herbst K, Boerner K, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med. 2020;12(556).
Dao Thi, V. L., Herbst, K., Boerner, K., Meurer, M., Kremer, L. P., Kirrmaier, D., Freistaedter, A., Papagiannidis, D., Galmozzi, C., Stanifer, M. L., Boulant, S., Klein, S., Chlanda, P., Khalid, D., Barreto Miranda, I., Schnitzler, P., Kräusslich, H. G., Knop, M., & Anders, S. (2020). A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Science Translational Medicine, 12(556). https://doi.org/10.1126/scitranslmed.abc7075
Dao Thi VL, et al. A Colorimetric RT-LAMP Assay and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples. Sci Transl Med. 2020 08 12;12(556) PubMed PMID: 32719001.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. AU - Dao Thi,Viet Loan, AU - Herbst,Konrad, AU - Boerner,Kathleen, AU - Meurer,Matthias, AU - Kremer,Lukas Pm, AU - Kirrmaier,Daniel, AU - Freistaedter,Andrew, AU - Papagiannidis,Dimitrios, AU - Galmozzi,Carla, AU - Stanifer,Megan L, AU - Boulant,Steeve, AU - Klein,Steffen, AU - Chlanda,Petr, AU - Khalid,Dina, AU - Barreto Miranda,Isabel, AU - Schnitzler,Paul, AU - Kräusslich,Hans-Georg, AU - Knop,Michael, AU - Anders,Simon, Y1 - 2020/07/27/ PY - 2020/05/08/received PY - 2020/07/23/accepted PY - 2020/7/29/pubmed PY - 2020/8/28/medline PY - 2020/7/29/entrez JF - Science translational medicine JO - Sci Transl Med VL - 12 IS - 556 N2 - The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions. SN - 1946-6242 UR - https://www.unboundmedicine.com/medline/citation/32719001/A_colorimetric_RT_LAMP_assay_and_LAMP_sequencing_for_detecting_SARS_CoV_2_RNA_in_clinical_samples_ L2 - http://stm.sciencemag.org/cgi/pmidlookup?view=short&amp;pmid=32719001 DB - PRIME DP - Unbound Medicine ER -