A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples

Sci Transl Med. 2020 Aug 12;12(556):eabc7075. doi: 10.1126/scitranslmed.abc7075. Epub 2020 Jul 27.

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.

Publication types

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

MeSH terms

  • Betacoronavirus / genetics*
  • Betacoronavirus / isolation & purification*
  • COVID-19
  • Colorimetry / methods
  • Colorimetry / statistics & numerical data
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / epidemiology
  • Coronavirus Infections / virology*
  • Coronavirus Nucleocapsid Proteins
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Molecular Diagnostic Techniques / statistics & numerical data
  • Nucleic Acid Amplification Techniques / methods*
  • Nucleic Acid Amplification Techniques / statistics & numerical data
  • Nucleocapsid Proteins / genetics
  • Pandemics
  • Phosphoproteins
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / epidemiology
  • Pneumonia, Viral / virology*
  • RNA, Viral / genetics
  • RNA, Viral / isolation & purification
  • RNA-Seq
  • SARS-CoV-2
  • Sensitivity and Specificity
  • Translational Research, Biomedical

Substances

  • Coronavirus Nucleocapsid Proteins
  • Nucleocapsid Proteins
  • Phosphoproteins
  • RNA, Viral
  • nucleocapsid phosphoprotein, SARS-CoV-2

Supplementary concepts

  • LAMP assay