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Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2
We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 ki...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998296/ https://www.ncbi.nlm.nih.gov/pubmed/33807908 http://dx.doi.org/10.3390/v13030420 |
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author | Curti, Lucía Ana Primost, Ivana Valla, Sofia Ibañez Alegre, Daiana Olguin Perglione, Cecilia Repizo, Guillermo Daniel Lara, Julia Parcerisa, Ivana Palacios, Antonela Llases, María Eugenia Rinflerch, Adriana Barrios, Melanie Pereyra Bonnet, Federico Gimenez, Carla Alejandra Marcone, Débora Natalia |
author_facet | Curti, Lucía Ana Primost, Ivana Valla, Sofia Ibañez Alegre, Daiana Olguin Perglione, Cecilia Repizo, Guillermo Daniel Lara, Julia Parcerisa, Ivana Palacios, Antonela Llases, María Eugenia Rinflerch, Adriana Barrios, Melanie Pereyra Bonnet, Federico Gimenez, Carla Alejandra Marcone, Débora Natalia |
author_sort | Curti, Lucía Ana |
collection | PubMed |
description | We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories. |
format | Online Article Text |
id | pubmed-7998296 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-79982962021-03-28 Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 Curti, Lucía Ana Primost, Ivana Valla, Sofia Ibañez Alegre, Daiana Olguin Perglione, Cecilia Repizo, Guillermo Daniel Lara, Julia Parcerisa, Ivana Palacios, Antonela Llases, María Eugenia Rinflerch, Adriana Barrios, Melanie Pereyra Bonnet, Federico Gimenez, Carla Alejandra Marcone, Débora Natalia Viruses Article We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories. MDPI 2021-03-05 /pmc/articles/PMC7998296/ /pubmed/33807908 http://dx.doi.org/10.3390/v13030420 Text en © 2021 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 (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ). |
spellingShingle | Article Curti, Lucía Ana Primost, Ivana Valla, Sofia Ibañez Alegre, Daiana Olguin Perglione, Cecilia Repizo, Guillermo Daniel Lara, Julia Parcerisa, Ivana Palacios, Antonela Llases, María Eugenia Rinflerch, Adriana Barrios, Melanie Pereyra Bonnet, Federico Gimenez, Carla Alejandra Marcone, Débora Natalia Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 |
title | Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 |
title_full | Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 |
title_fullStr | Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 |
title_full_unstemmed | Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 |
title_short | Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2 |
title_sort | evaluation of a lyophilized crispr-cas12 assay for a sensitive, specific, and rapid detection of sars-cov-2 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7998296/ https://www.ncbi.nlm.nih.gov/pubmed/33807908 http://dx.doi.org/10.3390/v13030420 |
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