RT Journal Article
SR Electronic
T1 A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples
JF Science Translational Medicine
FD American Association for the Advancement of Science
SP eabc7075
DO 10.1126/scitranslmed.abc7075
VO 12
IS 556
A1 Dao Thi, Viet Loan
A1 Herbst, Konrad
A1 Boerner, Kathleen
A1 Meurer, Matthias
A1 Kremer, Lukas PM
A1 Kirrmaier, Daniel
A1 Freistaedter, Andrew
A1 Papagiannidis, Dimitrios
A1 Galmozzi, Carla
A1 Stanifer, Megan L.
A1 Boulant, Steeve
A1 Klein, Steffen
A1 Chlanda, Petr
A1 Khalid, Dina
A1 Barreto Miranda, Isabel
A1 Schnitzler, Paul
A1 Kräusslich, Hans-Georg
A1 Knop, Michael
A1 Anders, Simon
YR 2020
UL http://stm.sciencemag.org/content/12/556/eabc7075.abstract
AB We need simple methods to rapidly test large numbers of people for infection with the SARS-CoV-2 coronavirus. Quantitative PCR (qPCR) after reverse transcription (RT), the standard method, is very sensitive but requires expensive instrumentation. Loop-mediated isothermal amplification (LAMP) is an alternative to qPCR that is faster and requires fewer resources. Dao Thi et al. tested the RT-LAMP assay on several hundred clinical RNA samples isolated from pharyngeal swabs collected from individuals being tested for COVID-19. They confirmed that the RT-LAMP assay was a simpler albeit less sensitive option compared to RT-qPCR for large-scale testing for SARS-CoV-2 RNA. These investigators also developed a simplified version of this method (direct swab–to–RT-LAMP assay) that did not require a prior RNA isolation step as well as a method for highly multiplexed sequencing of RT-LAMP reactions (LAMP-sequencing).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.