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Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

BACKGROUND: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique stil...

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Autores principales: de Oliveira Coelho, Bruna, Sanchuki, Heloisa Bruna Soligo, Zanette, Dalila Luciola, Nardin, Jeanine Marie, Morales, Hugo Manuel Paz, Fornazari, Bruna, Aoki, Mateus Nóbrega, Blanes, Lucas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996115/
https://www.ncbi.nlm.nih.gov/pubmed/33771097
http://dx.doi.org/10.1186/s10020-021-00289-0
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author de Oliveira Coelho, Bruna
Sanchuki, Heloisa Bruna Soligo
Zanette, Dalila Luciola
Nardin, Jeanine Marie
Morales, Hugo Manuel Paz
Fornazari, Bruna
Aoki, Mateus Nóbrega
Blanes, Lucas
author_facet de Oliveira Coelho, Bruna
Sanchuki, Heloisa Bruna Soligo
Zanette, Dalila Luciola
Nardin, Jeanine Marie
Morales, Hugo Manuel Paz
Fornazari, Bruna
Aoki, Mateus Nóbrega
Blanes, Lucas
author_sort de Oliveira Coelho, Bruna
collection PubMed
description BACKGROUND: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. METHODS: To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. RESULTS: First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. CONCLUSION: This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s10020-021-00289-0.
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spelling pubmed-79961152021-03-29 Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis de Oliveira Coelho, Bruna Sanchuki, Heloisa Bruna Soligo Zanette, Dalila Luciola Nardin, Jeanine Marie Morales, Hugo Manuel Paz Fornazari, Bruna Aoki, Mateus Nóbrega Blanes, Lucas Mol Med Research Article BACKGROUND: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. METHODS: To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. RESULTS: First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. CONCLUSION: This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s10020-021-00289-0. BioMed Central 2021-03-26 /pmc/articles/PMC7996115/ /pubmed/33771097 http://dx.doi.org/10.1186/s10020-021-00289-0 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Research Article
de Oliveira Coelho, Bruna
Sanchuki, Heloisa Bruna Soligo
Zanette, Dalila Luciola
Nardin, Jeanine Marie
Morales, Hugo Manuel Paz
Fornazari, Bruna
Aoki, Mateus Nóbrega
Blanes, Lucas
Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis
title Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis
title_full Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis
title_fullStr Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis
title_full_unstemmed Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis
title_short Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis
title_sort essential properties and pitfalls of colorimetric reverse transcription loop-mediated isothermal amplification as a point-of-care test for sars-cov-2 diagnosis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996115/
https://www.ncbi.nlm.nih.gov/pubmed/33771097
http://dx.doi.org/10.1186/s10020-021-00289-0
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