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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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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.
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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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