A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples
| العنوان: | 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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| المؤلفون: | Helena Sofia S. Teles, Américo G. Duarte, Ana Margarida Henriques, Wilson Antunes, Cristiana C. Santos, Catarina Pimentel, Catarina Amaral, Manuel A. Ramalho da Silva, Mónica Serrano, Elin Moe, Ricardo Vieira, Luís M. P. Lima, Marisa S. Reis, Gonçalo S. Afonso, M. Rita Ventura, Inês Gomes, Miguel Fevereiro |
| المساهمون: | Instituto de Tecnologia Química e Biológica António Xavier (ITQB) |
| المصدر: | Scientific Reports Scientific Reports, Vol 11, Iss 1, Pp 1-12 (2021) |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | Test strategy, Saliva, Molecular biology, Science, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Sensitivity and Specificity, Article, Colorimetry (chemical method), COVID-19 Testing, SDG 3 - Good Health and Well-being, Humans, General, Pandemics, Multidisciplinary, Chromatography, Portugal, SARS-CoV-2, Chemistry, Infectious-disease diagnostics, COVID-19, RNA, Nucleic acid amplification technique, Molecular Diagnostic Techniques, Medicine, RNA, Viral, Colorimetry, RNA extraction, Nucleic Acid Amplification Techniques, Viral load |
| الوصف: | Funding Information: We thank the members of the COVID-19 task force of ITQB NOVA and Mariana Pinho for helpful discussions and suggestions. In particular, we thank Claudio M. Soares and Adriano O. Henriques for their unconditional support and invaluable help in making this work possible. The authors acknowledge the assistance of Paula Chi-cau, ITQB NOVA, in the development of the saliva test and are grateful to Chuck Farah, São Paulo University, for the pGTf2 plasmid. This work was supported by (i) Project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020—“Programa Operacional Competitividade e Internacionalização” (POCI), (ii) the European Union’s Horizon 2020 research and innovation programme under grant agreement No 810856 and (iii) “Fundação para a Ciência e a Tecnologia” (FCT) through programme IF (IF/00124/2015) to C.P, (iv) through the project “DETECT: Development of an Easy, fast-Track and Economical Colorimetric Test for autonomous national diagnosis of COVID-19” Ref 433_613549914 (20/7/153), attributed to C.P., under the scope of the 2nd edition of the programme RESEARCH4COVID19 and partially supported by (v) the project “STOP-COVID—Strategies to prevent COVID-19 by early detection of asymptomatic carriers at increased risk: epidemiological studies and validation of a rapid in-house diagnostic test”, Ref 072559, funded by FEDER—“Fundo Europeu de Desenvolvimento Regional” from “Programa Opera-cional Regional Lisboa”. Publisher Copyright: © 2021, The Author(s). 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. publishersversion published |
| اللغة: | English |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7481371163952d02f203ad9b47d7964e https://hdl.handle.net/10362/142211 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....7481371163952d02f203ad9b47d7964e |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |