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Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2

At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maint...

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Autores principales: Ortiz-Cartagena, Concha, Fernández-García, Laura, Blasco, Lucia, Pacios, Olga, Bleriot, Inés, López, María, Cantón, Rafael, Tomás, María
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9604158/
https://www.ncbi.nlm.nih.gov/pubmed/36169448
http://dx.doi.org/10.1128/spectrum.02398-22
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author Ortiz-Cartagena, Concha
Fernández-García, Laura
Blasco, Lucia
Pacios, Olga
Bleriot, Inés
López, María
Cantón, Rafael
Tomás, María
author_facet Ortiz-Cartagena, Concha
Fernández-García, Laura
Blasco, Lucia
Pacios, Olga
Bleriot, Inés
López, María
Cantón, Rafael
Tomás, María
author_sort Ortiz-Cartagena, Concha
collection PubMed
description At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (C(T)) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples.
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spelling pubmed-96041582022-10-27 Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 Ortiz-Cartagena, Concha Fernández-García, Laura Blasco, Lucia Pacios, Olga Bleriot, Inés López, María Cantón, Rafael Tomás, María Microbiol Spectr Research Article At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (C(T)) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples. American Society for Microbiology 2022-09-28 /pmc/articles/PMC9604158/ /pubmed/36169448 http://dx.doi.org/10.1128/spectrum.02398-22 Text en Copyright © 2022 Ortiz-Cartagena et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Ortiz-Cartagena, Concha
Fernández-García, Laura
Blasco, Lucia
Pacios, Olga
Bleriot, Inés
López, María
Cantón, Rafael
Tomás, María
Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
title Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
title_full Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
title_fullStr Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
title_full_unstemmed Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
title_short Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
title_sort reverse transcription-loop-mediated isothermal amplification-crispr-cas13a technology as a promising diagnostic tool for sars-cov-2
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9604158/
https://www.ncbi.nlm.nih.gov/pubmed/36169448
http://dx.doi.org/10.1128/spectrum.02398-22
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