PLoS ONE (Jan 2023)

Starting from scratch: Step-by-step development of diagnostic tests for SARS-CoV-2 detection by RT-LAMP.

  • Diana Angélica Tapia-Sidas,
  • Brenda Yazmín Vargas-Hernández,
  • José Abrahán Ramírez-Pool,
  • Leandro Alberto Núñez-Muñoz,
  • Berenice Calderón-Pérez,
  • Rogelio González-González,
  • Luis Gabriel Brieba,
  • Rosalía Lira-Carmona,
  • Eduardo Ferat-Osorio,
  • Constantino López-Macías,
  • Roberto Ruiz-Medrano,
  • Beatriz Xoconostle-Cázares

DOI
https://doi.org/10.1371/journal.pone.0279681
Journal volume & issue
Vol. 18, no. 1
p. e0279681

Abstract

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The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people worldwide. Public health strategies to reduce viral transmission are based on widespread diagnostic testing to detect and isolate contagious patients. Several reverse transcription (RT)-PCR tests, along with other SARS-CoV-2 diagnostic assays, are available to attempt to cover the global demand. Loop-mediated isothermal amplification (LAMP) based methods have been established as rapid, accurate, point of care diagnostic tests for viral infections; hence, they represent an excellent alternative for SARS-CoV-2 detection. The aim of this study was to develop and describe molecular detection systems for SARS-CoV-2 based on RT-LAMP. Recombinant DNA polymerase from Bacillus stearothermophilus and thermostable engineered reverse transcriptase from Moloney Murine Leukemia Virus were expressed using a prokaryotic system and purified by fast protein liquid chromatography. These enzymes were used to set up fluorometric real time and colorimetric end-point RT-LAMP assays. Several reaction conditions were optimized such as reaction temperature, Tris-HCl concentration, and pH of the diagnostic tests. The key enzymes for RT-LAMP were purified and their enzymatic activity was determined. Standardized reaction conditions for both RT-LAMP assays were 65°C and a Tris-HCl-free buffer at pH 8.8. Colorimetric end-point RT-LAMP assay was successfully used for viral detection from clinical saliva samples with 100% sensitivity and 100% specificity compared to the results obtained by RT-qPCR based diagnostic protocols with Ct values until 30. The developed RT-LAMP diagnostic tests based on purified recombinant enzymes allowed a sensitive and specific detection of the nucleocapsid gene of SARS-CoV-2.