Actividad Investigadora
«An accurate amplification-free CRISPR/Cas12a-based assay for GES ß-lactamase detection».
Autores de IDIVAL
Autores ajenos al IDIVAL
- Ortiz-Cartagena C
- Pacios O
- Blasco L
- Tomás M
Unidades
Abstract
Guiana-Extended-Spectrum (GES) ß-lactamases belong to the minor class A ß-lactamases and are probably underdiagnosed due to a lack of specific diagnostic tests. There is therefore an urgent need to develop new molecular diagnostic tools that will be able to fill the gap in the detection of rare ß-lactamases. Here, we propose an optimized, amplification-free CRISPR/Cas12a-based assay for the accurate detection of GES ß-lactamases and we validate its application with clinical isolates (Graphic Abstract). Based on the results of examination of 79 standard collection, the proposed assay exhibited 100% sensitivity and specificity, as well as 100% positive and negative predictive values in less than 1.5 hours. We optimized the CRISPR/Cas12a method by harnessing a multiplex crRNA strategy, a highly efficient DNA reporter (TTATT-5C) and the Murine RNase Inhibitor to prevent crRNA degradation. This yielded limits of detection of 1ng/µL and 3ng/µL in Enterobacterales and Pseudomonas aeruginosa, respectively. The observed difference is due to the location of the bla(GES) gene. The gene occurs in a chromosomal integron present only in one to three copies in P. aeruginosa, whereas it occurs in plasmids present in multiple copies in Enterobacterales. The proposed method could be established as a routine diagnostic tool in clinical microbiology laboratories to fill the gap in availability of commercial diagnostic tests for GES ß-lactamases.
Copyright © 2025. Published by Elsevier Ltd.
Datos de la publicación
- ISSN/ISSNe:
- 0924-8579, 1872-7913
- Tipo:
- Article
- Páginas:
- 107506-107506
- PubMed:
- 40187665
International Journal of Antimicrobial Agents ELSEVIER
Documentos
- No hay documentos
Filiaciones
Keywords
- CRISPR; Cas12a; GES ß-lactamase; amplification-free
Campos de Estudio
Proyectos asociados
CB21/13/00068 Fariñas Álvarez, María Del Carmen
Investigador Principal: María del Carmen Fariñas Álvarez
CB21/13/00068 . INSTITUTO DE SALUD CARLOS III. . 2022
Cita
Pablo D, Fernández L, Rodríguez J, Fraile N, Ortiz C, Pacios O, García S, García S, Blasco L, Ocampo A, Calvo J, Tomás M. «An accurate amplification-free CRISPR/Cas12a-based assay for GES ß-lactamase detection». Int. J. Antimicrob. Agents. 2025. 66. (1):p. 107506-107506. IF:4,600. (1).
