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Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System
Background: The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and acc...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341061/ https://www.ncbi.nlm.nih.gov/pubmed/37443626 http://dx.doi.org/10.3390/diagnostics13132233 |
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author | de Sousa, Karoline Almeida Felix Nonaka, Carolina Kymie Vasques Khouri, Ricardo Gurgel Rocha, Clarissa Araújo Regis-Silva, Carlos Gustavo de Freitas Souza, Bruno Solano |
author_facet | de Sousa, Karoline Almeida Felix Nonaka, Carolina Kymie Vasques Khouri, Ricardo Gurgel Rocha, Clarissa Araújo Regis-Silva, Carlos Gustavo de Freitas Souza, Bruno Solano |
author_sort | de Sousa, Karoline Almeida Felix |
collection | PubMed |
description | Background: The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study. Methods: A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader. Results: The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL. Conclusion: The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays. |
format | Online Article Text |
id | pubmed-10341061 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-103410612023-07-14 Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System de Sousa, Karoline Almeida Felix Nonaka, Carolina Kymie Vasques Khouri, Ricardo Gurgel Rocha, Clarissa Araújo Regis-Silva, Carlos Gustavo de Freitas Souza, Bruno Solano Diagnostics (Basel) Communication Background: The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study. Methods: A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader. Results: The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL. Conclusion: The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays. MDPI 2023-06-30 /pmc/articles/PMC10341061/ /pubmed/37443626 http://dx.doi.org/10.3390/diagnostics13132233 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Communication de Sousa, Karoline Almeida Felix Nonaka, Carolina Kymie Vasques Khouri, Ricardo Gurgel Rocha, Clarissa Araújo Regis-Silva, Carlos Gustavo de Freitas Souza, Bruno Solano Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System |
title | Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System |
title_full | Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System |
title_fullStr | Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System |
title_full_unstemmed | Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System |
title_short | Rapid Detection of SARS-CoV-2 Based on the LAMP Assay Associated with the CRISPRCas12a System |
title_sort | rapid detection of sars-cov-2 based on the lamp assay associated with the crisprcas12a system |
topic | Communication |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10341061/ https://www.ncbi.nlm.nih.gov/pubmed/37443626 http://dx.doi.org/10.3390/diagnostics13132233 |
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