A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

Abstract Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testin...

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Autores principales:	Catarina Amaral, Wilson Antunes, Elin Moe, Américo G. Duarte, Luís M. P. Lima, Cristiana Santos, Inês L. Gomes, Gonçalo S. Afonso, Ricardo Vieira, Helena Sofia S. Teles, Marisa S. Reis, Manuel A. Ramalho da Silva, Ana Margarida Henriques, Miguel Fevereiro, M. Rita Ventura, Mónica Serrano, Catarina Pimentel
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/b004f05d7714426598f86c9447c7a6c3
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spelling	oai:doaj.org-article:b004f05d7714426598f86c9447c7a6c32021-12-02T18:50:53ZA molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples10.1038/s41598-021-95799-62045-2322https://doaj.org/article/b004f05d7714426598f86c9447c7a6c32021-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-95799-6https://doaj.org/toc/2045-2322Abstract Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.Catarina AmaralWilson AntunesElin MoeAmérico G. DuarteLuís M. P. LimaCristiana SantosInês L. GomesGonçalo S. AfonsoRicardo VieiraHelena Sofia S. TelesMarisa S. ReisManuel A. Ramalho da SilvaAna Margarida HenriquesMiguel FevereiroM. Rita VenturaMónica SerranoCatarina PimentelNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Catarina Amaral Wilson Antunes Elin Moe Américo G. Duarte Luís M. P. Lima Cristiana Santos Inês L. Gomes Gonçalo S. Afonso Ricardo Vieira Helena Sofia S. Teles Marisa S. Reis Manuel A. Ramalho da Silva Ana Margarida Henriques Miguel Fevereiro M. Rita Ventura Mónica Serrano Catarina Pimentel A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
description	Abstract Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.
format	article
author	Catarina Amaral Wilson Antunes Elin Moe Américo G. Duarte Luís M. P. Lima Cristiana Santos Inês L. Gomes Gonçalo S. Afonso Ricardo Vieira Helena Sofia S. Teles Marisa S. Reis Manuel A. Ramalho da Silva Ana Margarida Henriques Miguel Fevereiro M. Rita Ventura Mónica Serrano Catarina Pimentel
author_facet	Catarina Amaral Wilson Antunes Elin Moe Américo G. Duarte Luís M. P. Lima Cristiana Santos Inês L. Gomes Gonçalo S. Afonso Ricardo Vieira Helena Sofia S. Teles Marisa S. Reis Manuel A. Ramalho da Silva Ana Margarida Henriques Miguel Fevereiro M. Rita Ventura Mónica Serrano Catarina Pimentel
author_sort	Catarina Amaral
title	A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
title_short	A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
title_full	A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
title_fullStr	A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
title_full_unstemmed	A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
title_sort	molecular test based on rt-lamp for rapid, sensitive and inexpensive colorimetric detection of sars-cov-2 in clinical samples
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/b004f05d7714426598f86c9447c7a6c3
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A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

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