dc.contributor.author | Ortiz-Cartagena, Concha | |
dc.contributor.author | Pablo-Marcos, Daniel | |
dc.contributor.author | Fernández-García, Laura | |
dc.contributor.author | Blasco, Lucía | |
dc.contributor.author | Pacios, Olga | |
dc.contributor.author | Bleriot, Inés | |
dc.contributor.author | Siller, María | |
dc.contributor.author | López, María | |
dc.contributor.author | Fernández, Javier | |
dc.contributor.author | Aracil, Belen | |
dc.contributor.author | Fraile-Ribot, Pablo Arturo | |
dc.contributor.author | García-Fernández, Sergio | |
dc.contributor.author | Fernández-Cuenca, Felipe | |
dc.contributor.author | Hernández-García, Marta | |
dc.contributor.author | Cantón, Rafael | |
dc.contributor.author | Calvo-Montes, Jorge | |
dc.contributor.author | Tomás, María | |
dc.date.accessioned | 2023-07-24T10:53:55Z | |
dc.date.available | 2023-07-24T10:53:55Z | |
dc.date.issued | 2023 | |
dc.identifier.citation | Microbiol Spectr. 2023 Aug 17;11(4):e0132923. | es_ES |
dc.identifier.uri | http://hdl.handle.net/20.500.12105/16305 | |
dc.description.abstract | 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. | es_ES |
dc.description.sponsorship | This study was funded by grants PI19/00878 and PI22/00323 awarded to M. Tomás, within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research - European Regional Development Fund “A Way of Making Europe” and Instituto de Salud Carlos III FEDER, Spanish Networkfor the Research in Infectious Diseases (REIPI, RD16/0016/0006 and CIBER CB21/13/00012, CB21/13/00084 and CB21/13/00095), Instituto de Salud Carlos III FEDER. The research was also funded by grant IN607D 2021/10 within the GAIN (Agencia Gallega de Innovación) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/), and finally an ESCMID grant (European Society of Clinical Microbiology and Infectious Diseases) awarded to L. Fernández-García. O. Pacios, L. Fernández-García, and M. López were financially supported by the grants IN606A-2020/035, IN606B-2021/013 and IN606C-2022/002, respectively (GAIN, Xunta de Galicia). D. Pablo-Marcos was financially supported by a López Albo grant. I. Bleriot was financially supportedby pFIS pro-511 g (ISCIII, FI20/00302). We declare that there are no conflicts of interest. | es_ES |
dc.language.iso | eng | es_ES |
dc.publisher | American Society for Microbiology (ASM) | es_ES |
dc.type.hasVersion | VoR | es_ES |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | * |
dc.subject | Detection | es_ES |
dc.subject | GES | es_ES |
dc.subject | OXA-48 | es_ES |
dc.subject | LAMP | es_ES |
dc.subject | CRISPR-Cas13a | es_ES |
dc.title | CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases | es_ES |
dc.type | research article | es_ES |
dc.rights.license | Atribución 4.0 Internacional | * |
dc.identifier.pubmedID | 37466441 | es_ES |
dc.format.page | e0132923 | es_ES |
dc.identifier.doi | 10.1128/spectrum.01329-23 | es_ES |
dc.contributor.funder | Plan Nacional de I+D+i (España) | es_ES |
dc.contributor.funder | Instituto de Salud Carlos III | es_ES |
dc.contributor.funder | Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF) | es_ES |
dc.contributor.funder | RETICS-Investigación en Patología Infecciosa (REIPI-ISCIII) (España) | es_ES |
dc.contributor.funder | Xunta de Galicia (España) | es_ES |
dc.contributor.funder | European Society of Clinical Microbiology and Infectious Diseases | es_ES |
dc.contributor.funder | Centro de Investigación Biomédica en Red - CIBERES (Enfermedades Respiratorias) | es_ES |
dc.contributor.funder | Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas) | es_ES |
dc.description.peerreviewed | Sí | es_ES |
dc.identifier.e-issn | 2165-0497 | es_ES |
dc.relation.publisherversion | https://doi.org/10.1128/spectrum.01329-23 | es_ES |
dc.identifier.journal | Microbiology spectrum | es_ES |
dc.repisalud.centro | ISCIII::Centro Nacional de Microbiología | es_ES |
dc.repisalud.institucion | ISCIII | es_ES |
dc.rights.accessRights | open access | es_ES |
dc.relation.projectFECYT | info:eu-repo/grantAgreement/MINECO//RD16%2F0016%2F0006/ES/RED ESPAÑOLA DE INVESTIGACIÓN EN PATOLOGÍAS INFECCIOSAS/ | es_ES |
dc.relation.projectFIS | info:fis/Instituto de Salud Carlos III/Programa Estatal de Generación de Conocimiento y Fortalecimiento del Sistema Español de I+D+I/Subprograma Estatal de Generación de Conocimiento/PI19 - Proyectos de investigacion en salud (AES 2019). Modalidad proyectos en salud. (2019)/PI19/00878 | es_ES |
dc.relation.projectFIS | info:fis/Instituto de Salud Carlos III///PI22- Proyectos de I+D+I en salud (AES 2022). (2022)/PI22/00323 | es_ES |
dc.relation.projectFIS | info:eu-repo/grantAgreement/ES/FI20/00302 | es_ES |
dc.relation.projectFIS | info:eu-repo/grantAgreement/ES/CB21/13/00012 | es_ES |
dc.relation.projectFIS | info:eu-repo/grantAgreement/ES/CB21/13/00084 | es_ES |
dc.relation.projectFIS | info:eu-repo/grantAgreement/ES/CB21/13/00095 | es_ES |