{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T13:56:54Z","timestamp":1785938214468,"version":"3.56.0"},"reference-count":60,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T00:00:00Z","timestamp":1785888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan","award":["BR24992881"],"award-info":[{"award-number":["BR24992881"]}]},{"award":["BR24992881"],"award-info":[{"award-number":["BR24992881"]}],"id":[{"id":"https:\/\/ror.org\/05tyne317","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biosensors"],"abstract":"<jats:p>Acinetobacter baumannii is a critical WHO priority pathogen due to its multidrug resistance and high mortality in carbapenem-resistant infections. Resistance is predominantly mediated by class D carbapenemase genes blaOXA-23 and blaOXA-40, which spread rapidly via horizontal gene transfer in healthcare settings. To address the lack of a rapid assay capable of detecting both blaOXA-23 and blaOXA-40 in a single analytical workflow, we developed a multiplex two-step RPA\u2013CRISPR\/Cas12a assay. Since infections caused by strains harboring either gene require identical therapeutic management, their co-detection in a single reaction is clinically justified. Although simultaneous use of two crRNAs within a single CRISPR\/Cas12a reaction is often considered technically challenging due to potential inter-crRNA competition, here it advantageously enables dual-target coverage without compromising sensitivity. The assay demonstrated high specificity with no cross-reactivity against a panel of clinically relevant bacterial species, including closely related Acinetobacter spp. Evaluation using genomic DNA extracted from 63 cultured clinical A. baumannii isolates revealed blaOXA-23 in 19 isolates (30.2%), blaOXA-40 in 28 (44.4%), and co-carriage of both genes in 9 (14.3%), with at least one resistance gene detected in 60.3% of isolates. The complete workflow was accomplished within 45 min without specialized equipment, offering a rapid, sensitive, and cost-effective solution for point-of-care molecular surveillance of carbapenem-resistant A. baumannii in clinical and resource-limited settings.<\/jats:p>","DOI":"10.3390\/bios16080422","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T13:16:29Z","timestamp":1785935789000},"page":"422","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Multiplex RPA-CRISPR\/Cas12a Assay for Rapid Detection of Class D OXA-Type Carbapenem-Resistant Acinetobacter baumannii"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9847-2679","authenticated-orcid":false,"given":"Meruyert","family":"Amanzholova","sequence":"first","affiliation":[{"name":"National Center for Biotechnology, Astana 010000,  Kazakhstan"},{"name":"Faculty of Biology and Biotechnology, Farabi Kazakh National University, Almaty 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ainur","family":"Akimbekova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Astana 010000,  Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aisha","family":"Shaizadinova","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Astana 010000,  Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-7314-9153","authenticated-orcid":false,"given":"Nazgul","family":"Sutimbekova","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Virology,  Astana Medical University, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nelya","family":"Bissenova","sequence":"additional","affiliation":[{"name":"National Scientific Medical Center, Astana 010000, Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2075-307X","authenticated-orcid":false,"given":"Pavel","family":"Tarlykov","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Astana 010000,  Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6974-9138","authenticated-orcid":false,"given":"Sailau","family":"Abeldenov","sequence":"additional","affiliation":[{"name":"National Center for Biotechnology, Astana 010000,  Kazakhstan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mancuso, G., Midiri, A., Gerace, E., and Biondo, C. 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