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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...

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Autores principales: de Sousa, Karoline Almeida Felix, Nonaka, Carolina Kymie Vasques, Khouri, Ricardo, Gurgel Rocha, Clarissa Araújo, Regis-Silva, Carlos Gustavo, de Freitas Souza, Bruno Solano
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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.
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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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