{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,11]],"date-time":"2026-09-11T19:17:15Z","timestamp":1789154235880,"version":"build-2803163510"},"reference-count":42,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2025,3,29]],"date-time":"2025-03-29T00:00:00Z","timestamp":1743206400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science and Higher Education of the Russian Federation","award":["075-15-2019-1666"],"award-info":[{"award-number":["075-15-2019-1666"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>The blaOXA-1 gene encodes an oxacillin-hydrolyzing beta-lactamase of extended-spectrum beta-lactamase (ESBL)-producing microorganisms. The blaOXA-1 gene is found in the resistomes of some Enterobacteriaceae, Morganellaceae, Pasteurellaceae, Moraxellaceae, Aeromonadaceae, Pseudomonadaceae, Yersiniaceae, and Vibrionaceae. Most ESBL detection methods, including those to detect OXA-1-producing microorganisms, are time-consuming, and require specialized equipment and qualified personnel. Here, we report a new CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)\/Cas12a-based detection assay coupled with polymerase chain reaction (PCR) to sensitively detect OXA-1-bearing microorganisms. The PCR-coupled CRISPR\/Cas12a-based fluorescence assay includes (i) a pre-amplification step and (ii) a nucleic acid detection step. The pre-amplification step is based on a commonly used PCR, and the detection step is based on the CRISPR\/Cas12a property to nonspecifically hydrolyze single-stranded DNA fluorescent reporter molecules. The pre-amplification step takes 65 min, and the detection step is shortened and takes only 5 min. The developed assay can easily detect single (1.25) copies of the blaOXA-1 gene in a reaction and is efficient not only in the detection of a blaOXA-1 model matrix but also in the detection of blaOXA-1-positive microorganisms. We hope that our assay has the potential to improve the monitoring of OXA-1-producing microorganisms and therefore contribute to mitigating the deadly global threat of antibiotic-resistant microorganisms.<\/jats:p>","DOI":"10.3390\/cimb47040238","type":"journal-article","created":{"date-parts":[[2025,3,31]],"date-time":"2025-03-31T02:50:16Z","timestamp":1743389416000},"page":"238","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Ultrasensitive CRISPR\/Cas12a-Based System for Detection of BlaOXA-1 Gene in Antibiotic-Resistant Microorganisms"],"prefix":"10.3390","volume":"47","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3145-3702","authenticated-orcid":false,"given":"Marina","family":"Tyumentseva","sequence":"first","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0537-2586","authenticated-orcid":false,"given":"Aleksandr","family":"Tyumentsev","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Prelovskaya","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-4256-0435","authenticated-orcid":false,"given":"Andrey","family":"Akinin","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5646-538X","authenticated-orcid":false,"given":"Yulia","family":"Mikhailova","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7409-077X","authenticated-orcid":false,"given":"Andrey","family":"Shelenkov","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4721-3447","authenticated-orcid":false,"given":"Anna","family":"Panevina","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4228-9044","authenticated-orcid":false,"given":"Vasiliy","family":"Akimkin","sequence":"additional","affiliation":[{"name":"Central Research Institute of Epidemiology, Novogireevskaya Str., 3a, 111123 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,29]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2025, March 20). Antimicrobial Resistance. Available online: https:\/\/www.who.int\/news-room\/fact-sheets\/detail\/antimicrobial-resistance."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1038\/s41579-018-0098-9","article-title":"Innovative and Rapid Antimicrobial Susceptibility Testing Systems","volume":"17","author":"Bachmann","year":"2019","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","first-page":"e01307-18","article-title":"CRISPR-Cas Biology and Its Application to Infectious Diseases","volume":"59","author":"Strich","year":"2021","journal-title":"J. Clin. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1139\/cjm-2018-0275","article-title":"Horizontal Transfer of Antibiotic Resistance Genes in Clinical Environments","volume":"65","author":"Lerminiaux","year":"2019","journal-title":"Can. J. Microbiol."},{"key":"ref_5","unstructured":"O\u2019Neill, J. (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations, Wellcome Trust. Available online: https:\/\/amr-review.org\/."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1086\/604713","article-title":"A multinational survey of risk factors for infection with extended-spectrum beta-lactamase-producing Enterobacteriaceae in nonhospitalized patients","volume":"49","author":"Arslan","year":"2009","journal-title":"Clin. Infect. Dis."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Karaiskos, I., and Giamarellou, H. (2020). Carbapenem-sparing strategies for ESBL producers: When and how. Antibiotics, 9.","DOI":"10.3390\/antibiotics9020061"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"7378","DOI":"10.1073\/pnas.84.21.7378","article-title":"Precise insertion of antibiotic resistance determinants into Tn21-like transposons: Nucleotide sequence of the OXA-1 beta-lactamase gene","volume":"84","author":"Ouellette","year":"1987","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1128\/CMR.14.4.933-951.2001","article-title":"Extended-spectrum beta-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat","volume":"14","author":"Bradford","year":"2001","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1128\/CMR.18.4.657-686.2005","article-title":"Extended-spectrum beta-lactamases: A clinical update","volume":"18","author":"Paterson","year":"2005","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1167","DOI":"10.1128\/JCM.01988-06","article-title":"Detection of extended-spectrum beta-lactamases among Enterobacteriaceae by use of semiautomated microbiology systems and manual detection procedures","volume":"45","author":"Wiegand","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Tyumentseva, M., Tyumentsev, A., and Akimkin, V. (2023). CRISPR\/Cas9 landscape: Current state and future perspectives. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms242216077"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1126\/science.aam9321","article-title":"Nucleic acid detection with CRISPR-Cas13a\/C2c2","volume":"356","author":"Gootenberg","year":"2017","journal-title":"Science"},{"key":"ref_15","unstructured":"Akimkin, V.G. (2020). Genetic Technologies, Central Research Institute for Epidemiology."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Lv, H., Wang, J., Zhang, J., Chen, Y., Yin, L., Jin, D., Gu, D., Zhao, H., Xu, Y., and Wang, J. (2021). Definition of CRISPR Cas12a trans-cleavage units to facilitate CRISPR diagnostics. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.766464"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"463","DOI":"10.1007\/s13206-022-00081-0","article-title":"Highly efficient DNA reporter for CRISPR\/Cas12a-based specific and sensitive biosensor","volume":"16","author":"Lee","year":"2022","journal-title":"Biochip J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"8377","DOI":"10.1093\/nar\/gkac578","article-title":"Enhancement of CRISPR\/Cas12a trans-cleavage activity using hairpin DNA reporters","volume":"50","author":"Rossetti","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Tyumentseva, M., Mikhaylova, Y., Prelovskaya, A., Karbyshev, K., Tyumentsev, A., Petrova, L., Mironova, A., Zamyatin, M., Shelenkov, A., and Akimkin, V. (2021). CRISPR element patterns vs. pathoadaptability of clinical Pseudomonas aeruginosa isolates from a medical center in Moscow, Russia. Antibiotics, 10.","DOI":"10.3390\/antibiotics10111301"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/B978-0-12-801185-0.00001-5","article-title":"In vitro enzymology of Cas9","volume":"Volume 546","author":"Anders","year":"2014","journal-title":"Methods in Enzymology"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Shin, J., Kim, S.R., Xie, Z., Jin, Y.-S., and Wang, Y.-C. (2024). A CRISPR\/Cas12a-based system for sensitive detection of antimicrobial-resistant genes in carbapenem-resistant Enterobacterales. Biosensors, 14.","DOI":"10.3390\/bios14040194"},{"key":"ref_23","unstructured":"Akimkin, V.G., Tiumentsev, A.I., and Tiumentseva, M.A. (2021). Crispr\/Cas System for Detecting Proviral HIV DNA. (WO2021118409A1), World Patent."},{"key":"ref_24","unstructured":"Akimkin, V.G., Tiumentsev, A.I., and Tiumentseva, M.A. (2021). Crispr\/Cas System for Detecting an Antibiotic Resistance Gene. (WO2021211012A1), World Patent."},{"key":"ref_25","unstructured":"Tiumentsev, A.I., Tiumentseva, M.A., Akimkin, V.G., and Prelovskaya, A.N. (2023). Crispr-cas14 System for Detecting SARS-CoV-2 Virus RNA at Ultra-Low Concentrations. (WO2023055255A1), World Patent."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"13005","DOI":"10.3892\/mmr.2023.13005","article-title":"CRISPR-based diagnostic approaches: Implications for rapid management of future pandemics (Review)","volume":"27","author":"Shariq","year":"2023","journal-title":"Mol. Med. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/S0378-1097(03)00306-9","article-title":"Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae","volume":"223","author":"Colom","year":"2003","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1038\/ja.2015.68","article-title":"Development of a multiplex PCR system and its application in detection of blaSHV, blaTEM, blaCTX-M-1, blaCTX-M-9 and blaOXA-1 group genes in clinical Klebsiella pneumoniae and Escherichia coli strains","volume":"68","author":"Ogutu","year":"2015","journal-title":"J. Antibiot."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Probst, K., Boutin, S., Bandilla, M., Heeg, K., and Dalpke, A.H. (2021). Fast and automated detection of common carbapenemase genes using multiplex real-time PCR on the BD MAXTM system. J. Microbiol. Methods, 185.","DOI":"10.1016\/j.mimet.2021.106224"},{"key":"ref_30","unstructured":"Cohen, S.S. (1997). A Guide to Polyamines, Oxford University Press."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2687","DOI":"10.1016\/S0021-9258(18)34978-0","article-title":"Catenation of DNA rings by topoisomerases. Mechanism of control by spermidine","volume":"257","author":"Krasnow","year":"1982","journal-title":"J. Biol. Chem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"11311","DOI":"10.1074\/jbc.RA119.007619","article-title":"Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1-Cas2 integrase","volume":"294","author":"Plateau","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1615","DOI":"10.1038\/ncomms2620","article-title":"Impact of macromolecular crowding on DNA replication","volume":"4","author":"Akabayov","year":"2013","journal-title":"Nat. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"10577","DOI":"10.1074\/jbc.R100005200","article-title":"The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media","volume":"276","author":"Minton","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1146\/annurev.bb.22.060193.000331","article-title":"Macromolecular crowding: Biochemical, biophysical, and physiological consequences","volume":"22","author":"Zimmerman","year":"1993","journal-title":"Annu. Rev. Biophys. Biomol. Struct."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Wang, Q., Liang, K.-C., Czader, A., Waxham, M.N., and Cheung, M.S. (2011). The effect of macromolecular crowding, ionic strength and calcium binding on calmodulin dynamics. PLoS Comput. Biol., 7.","DOI":"10.1371\/journal.pcbi.1002114"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3870","DOI":"10.1093\/emboj\/19.15.3870","article-title":"Macromolecular crowding perturbs protein refolding kinetics: Implications for folding inside the cell","volume":"19","author":"Wain","year":"2000","journal-title":"EMBO J."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1016\/0022-2836(91)90595-W","article-title":"Complementary recognition in condensed DNA: Accelerated DNA renaturation","volume":"222","author":"Sikorav","year":"1991","journal-title":"J. Mol. Biol."},{"key":"ref_39","unstructured":"Akimkin, V.G., Tiumentsev, A.I., Tiumentseva, M.A., and Shagin, D.A. (2020). Method for Producing a Preparation of Highly-Purified Recombinant Cas Nuclease. (WO2020197436A1), World Patent."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1007\/s41664-022-00212-4","article-title":"Improved strategies for CRISPR-Cas12-based nucleic acids detection","volume":"6","author":"Qiu","year":"2022","journal-title":"J. Anal. Test."},{"key":"ref_41","first-page":"1","article-title":"Glycine as a stabilizing osmolyte for Renilla luciferase: A kinetic and molecular dynamics analysis","volume":"43","author":"Salehian","year":"2024","journal-title":"Biocatal. Biotransform."},{"key":"ref_42","first-page":"114","article-title":"Optimization of a method for detecting single copies of hepatitis B virus DNA using CRISPR\/Cas systems","volume":"23","author":"Tyumentseva","year":"2025","journal-title":"Epidemiol. Vakcinoprofil."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/4\/238\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T17:05:03Z","timestamp":1760029503000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/47\/4\/238"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,29]]},"references-count":42,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2025,4]]}},"alternative-id":["cimb47040238"],"URL":"https:\/\/doi.org\/10.3390\/cimb47040238","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,3,29]]}}}