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CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases
Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10434040/ https://www.ncbi.nlm.nih.gov/pubmed/37466441 http://dx.doi.org/10.1128/spectrum.01329-23 |
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author | Ortiz-Cartagena, Concha Pablo-Marcos, Daniel Fernández-García, Laura Blasco, Lucía Pacios, Olga Bleriot, Inés Siller, María López, María Fernández, Javier Aracil, Belén Fraile-Ribot, Pablo Arturo García-Fernández, Sergio Fernández-Cuenca, Felipe Hernández-García, Marta Cantón, Rafael Calvo-Montes, Jorge Tomás, María |
author_facet | Ortiz-Cartagena, Concha Pablo-Marcos, Daniel Fernández-García, Laura Blasco, Lucía Pacios, Olga Bleriot, Inés Siller, María López, María Fernández, Javier Aracil, Belén Fraile-Ribot, Pablo Arturo García-Fernández, Sergio Fernández-Cuenca, Felipe Hernández-García, Marta Cantón, Rafael Calvo-Montes, Jorge Tomás, María |
author_sort | Ortiz-Cartagena, Concha |
collection | PubMed |
description | Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction. |
format | Online Article Text |
id | pubmed-10434040 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-104340402023-08-18 CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases Ortiz-Cartagena, Concha Pablo-Marcos, Daniel Fernández-García, Laura Blasco, Lucía Pacios, Olga Bleriot, Inés Siller, María López, María Fernández, Javier Aracil, Belén Fraile-Ribot, Pablo Arturo García-Fernández, Sergio Fernández-Cuenca, Felipe Hernández-García, Marta Cantón, Rafael Calvo-Montes, Jorge Tomás, María Microbiol Spectr Research Article Carbapenem-resistant pathogens have been recognized as a health concern as they are both difficult to treat and detect in clinical microbiology laboratories. Researchers are making great efforts to develop highly specific, sensitive, accurate, and rapid diagnostic techniques, required to prevent the spread of these microorganisms and improve the prognosis of patients. In this context, CRISPR-Cas systems are proposed as promising tools for the development of diagnostic methods due to their high specificity; the Cas13a endonuclease can discriminate single nucleotide changes and displays collateral cleavage activity against single-stranded RNA molecules when activated. This technology is usually combined with isothermal pre-amplification reactions in order to increase its sensitivity. We have developed a new LAMP-CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for nucleic acid purification and concentration. To evaluate the assay, we used 68 OXA-48-like-producing Klebsiella pneumoniae clinical isolates as well as 64 Enterobacter cloacae complex GES-6, 14 Pseudomonas aeruginosa GES-5, 9 Serratia marcescens GES-6, 5 P. aeruginosa GES-6, and 3 P. aeruginosa (GES-15, GES-27, and GES-40) and 1 K. pneumoniae GES-2 isolates. The assay, which takes less than 2 h and costs approximately 10 € per reaction, exhibited 100% specificity and sensitivity (99% confidence interval [CI]) for both OXA-48 and all GES carbapenemases. IMPORTANCE Carbapenems are one of the last-resort antibiotics for defense against multidrug-resistant pathogens. Multiple nucleic acid amplification methods, including multiplex PCR, multiplex loop-mediated isothermal amplification (LAMP) and multiplex RPAs, can achieve rapid, accurate, and simultaneous detection of several resistance genes to carbapenems in a single reaction. However, these assays need thermal cycling steps and specialized instruments, giving them limited application in the field. In this work, we adapted with high specificity and sensitivity values, a new LAMP CRISPR-Cas13a-based assay for the detection of OXA-48 and GES carbapenemases in clinical samples without the need for RNA extraction. American Society for Microbiology 2023-07-19 /pmc/articles/PMC10434040/ /pubmed/37466441 http://dx.doi.org/10.1128/spectrum.01329-23 Text en Copyright © 2023 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 Pablo-Marcos, Daniel Fernández-García, Laura Blasco, Lucía Pacios, Olga Bleriot, Inés Siller, María López, María Fernández, Javier Aracil, Belén Fraile-Ribot, Pablo Arturo García-Fernández, Sergio Fernández-Cuenca, Felipe Hernández-García, Marta Cantón, Rafael Calvo-Montes, Jorge Tomás, María CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases |
title | CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases |
title_full | CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases |
title_fullStr | CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases |
title_full_unstemmed | CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases |
title_short | CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases |
title_sort | crispr-cas13a-based assay for accurate detection of oxa-48 and ges carbapenemases |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10434040/ https://www.ncbi.nlm.nih.gov/pubmed/37466441 http://dx.doi.org/10.1128/spectrum.01329-23 |
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