| Autor: |
Dao Thi, Viet Loan, Herbst, Konrad, Boerner, Kathleen, Meurer, Matthias, Kremer, Lukas PM, Kirrmaier, Daniel, Freistaedter, Andrew, Papagiannidis, Dimitrios, Galmozzi, Carla, Stanifer, Megan L., Boulant, Steeve, Klein, Steffen, Chlanda, Petr, Khalid, Dina, Barreto Miranda, Isabel, Schnitzler, Paul, Kräusslich, Hans-Georg, Knop, Michael, Anders, Simon |
| Předmět: |
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| Zdroj: |
Science Translational Medicine; 8/12/2020, Vol. 12 Issue 556, p1-13, 13p |
| Abstrakt: |
A colorful test for SARS-CoV-2 RNA detection: 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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