2026-06-14T02:28:43Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/163052024-11-28T16:50:34Zcom_20.500.12105_2052com_20.500.12105_2051com_20.500.12105_15322col_20.500.12105_19609col_20.500.12105_16958col_20.500.12105_16967col_20.500.12105_16970col_20.500.12105_16985col_20.500.12105_16988col_20.500.12105_16990

00925njm 22002777a 4500 dc Ortiz-Cartagena, Concha author Pablo-Marcos, Daniel author Fernández-García, Laura author Blasco, Lucía author Pacios, Olga author Bleriot, Inés author Siller, María author López, María author Fernández, Javier author Aracil, Belen author Fraile-Ribot, Pablo Arturo author García-Fernández, Sergio author Fernández-Cuenca, Felipe author Hernández-García, Marta author Cantón, Rafael author Calvo-Montes, Jorge author Tomás, María author 2023 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. Microbiol Spectr. 2023 Aug 17;11(4):e0132923. 10.1128/spectrum.01329-23 2165-0497 Microbiology spectrum 37466441 http://hdl.handle.net/20.500.12105/16305 Detection GES OXA-48 LAMP CRISPR-Cas13a CRISPR-Cas13a-Based Assay for Accurate Detection of OXA-48 and GES Carbapenemases