tetano
Editor, Senior Moderator
Sci Transl Med
. 2020 Jul 27;eabc7075.
doi: 10.1126/scitranslmed.abc7075. Online ahead of print.
A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples
Viet Loan Dao Thi[SUP] 1 2 [/SUP], Konrad Herbst[SUP] #[/SUP][SUP] 3 [/SUP], Kathleen Boerner[SUP] #[/SUP][SUP] 2 4 [/SUP], Matthias Meurer[SUP] #[/SUP][SUP] 3 [/SUP], Lukas Pm Kremer[SUP] 3 5 6 [/SUP], Daniel Kirrmaier[SUP] 3 5 [/SUP], Andrew Freistaedter[SUP] 7 2 [/SUP], Dimitrios Papagiannidis[SUP] 3 [/SUP], Carla Galmozzi[SUP] 3 6 [/SUP], Megan L Stanifer[SUP] 2 [/SUP], Steeve Boulant[SUP] 2 5 [/SUP], Steffen Klein[SUP] 7 2 [/SUP], Petr Chlanda[SUP] 7 2 [/SUP], Dina Khalid[SUP] 2 [/SUP], Isabel Barreto Miranda[SUP] 2 [/SUP], Paul Schnitzler[SUP] 2 [/SUP], Hans-Georg Kr?usslich[SUP] 2 4 [/SUP], Michael Knop[SUP] 8 5 6 [/SUP], Simon Anders[SUP] 8 [/SUP]
Affiliations
Abstract
The COVID-19 pandemic caused by the SARS-CoV-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-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.
. 2020 Jul 27;eabc7075.
doi: 10.1126/scitranslmed.abc7075. Online ahead of print.
A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples
Viet Loan Dao Thi[SUP] 1 2 [/SUP], Konrad Herbst[SUP] #[/SUP][SUP] 3 [/SUP], Kathleen Boerner[SUP] #[/SUP][SUP] 2 4 [/SUP], Matthias Meurer[SUP] #[/SUP][SUP] 3 [/SUP], Lukas Pm Kremer[SUP] 3 5 6 [/SUP], Daniel Kirrmaier[SUP] 3 5 [/SUP], Andrew Freistaedter[SUP] 7 2 [/SUP], Dimitrios Papagiannidis[SUP] 3 [/SUP], Carla Galmozzi[SUP] 3 6 [/SUP], Megan L Stanifer[SUP] 2 [/SUP], Steeve Boulant[SUP] 2 5 [/SUP], Steffen Klein[SUP] 7 2 [/SUP], Petr Chlanda[SUP] 7 2 [/SUP], Dina Khalid[SUP] 2 [/SUP], Isabel Barreto Miranda[SUP] 2 [/SUP], Paul Schnitzler[SUP] 2 [/SUP], Hans-Georg Kr?usslich[SUP] 2 4 [/SUP], Michael Knop[SUP] 8 5 6 [/SUP], Simon Anders[SUP] 8 [/SUP]
Affiliations
- PMID: 32719001
- DOI: 10.1126/scitranslmed.abc7075
Abstract
The COVID-19 pandemic caused by the SARS-CoV-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-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.