{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T02:26:54Z","timestamp":1789698414600,"version":"4.0.1"},"reference-count":39,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T00:00:00Z","timestamp":1755561600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Russian Federation","award":["122030100170-5"],"award-info":[{"award-number":["122030100170-5"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>A straightforward approach is suggested to selectively recognize specific products of loop-mediated isothermal amplification (LAMP) with the Cas12a nuclease without a need for a protospacer adjacent motif (PAM) in the sequence of LAMP amplicons (LAMPlicons). This strategy is based on the presence of single-stranded DNA loops in LAMPlicons and the ability of Cas12a to be trans-activated via the binding of guide RNA (gRNA) to single-stranded DNA in the absence of PAM. The approach feasibility is demonstrated on Clavibacter species\u2014multiple bacterial plant pathogens that cause harmful diseases in agriculturally important plants. For Clavibacter species, the detection sensitivity of the developed PAM-independent LAMP\/Cas12a system was determined by that of LAMP. The overall detection selectivity was enhanced by the Cas12a analysis of LAMPlicons. It was shown that the LAMP\/Cas12a detection system can be fine-tuned by carefully designing gRNA to selectively distinguish C. sepedonicus from other Clavibacter species based on single nucleotide substitutions in the targeted LAMPlicon loop. The suggested loop-based Cas12a analysis of LAMPlicons was compatible with the format of a single test tube assay with the option of naked-eye detection. The findings broaden the palette of approaches to designing PAM-independent LAMP\/Cas12a detection systems with potential for on-site testing.<\/jats:p>","DOI":"10.3390\/ijms26168014","type":"journal-article","created":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T15:29:29Z","timestamp":1755617369000},"page":"8014","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["PAM-Independent Cas12a Detection of Specific LAMP Products by Targeting Amplicon Loops"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3197-6415","authenticated-orcid":false,"given":"Konstantin G.","family":"Ptitsyn","sequence":"first","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonid K.","family":"Kurbatov","sequence":"additional","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6224-4041","authenticated-orcid":false,"given":"Svetlana A.","family":"Khmeleva","sequence":"additional","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-2962-6152","authenticated-orcid":false,"given":"Daria D.","family":"Morozova","sequence":"additional","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olga S.","family":"Timoshenko","sequence":"additional","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2863-8562","authenticated-orcid":false,"given":"Elena V.","family":"Suprun","sequence":"additional","affiliation":[{"name":"Chemistry Faculty, M.V. Lomonosov Moscow State University, 1\/3 Lenin Hills, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0519-1745","authenticated-orcid":false,"given":"Sergey P.","family":"Radko","sequence":"additional","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrey V.","family":"Lisitsa","sequence":"additional","affiliation":[{"name":"V.N. Orekhovich Institute of Biomedical Chemistry, 10 Pogodinskaya St., 119121 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"104","DOI":"10.3892\/mmr.2023.12991","article-title":"Advances in the application of molecular diagnostic techniques for the detection of infectious disease pathogens (Review)","volume":"27","author":"Liu","year":"2023","journal-title":"Mol. Med. Rep."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Hansen, S., and Abd El Wahed, A. (2020). Point-Of-Care or Point-Of-Need Diagnostic Tests: Time to Change Outbreak Investigation and Pathogen Detection. Trop. Med. Infect. Dis., 5.","DOI":"10.3390\/tropicalmed5040151"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1177\/2472630320962003","article-title":"Point-of-Need Diagnostics for Foodborne Pathogen Screening","volume":"26","author":"Campbell","year":"2021","journal-title":"SLAS Technol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Lee, S.H., Park, S., Kim, B.N., Kwon, O.S., Rho, W.-Y., and Jun, B.-H. (2019). Emerging ultrafast nucleic acid amplification technologies for next-generation molecular diagnostics. Biosens. Bioelectron., 141.","DOI":"10.1016\/j.bios.2019.111448"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Yang, N., Zhang, H., Han, X., Liu, Z., and Lu, Y. (2024). Advancements and applications of loop-mediated isothermal amplification technology: A comprehensive overview. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1406632"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Atceken, N., Yigci, D., Ozdalgic, B., and Tasoglu, S. (2022). CRISPR-Cas-Integrated LAMP. Biosensors, 12.","DOI":"10.3390\/bios12111035"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"118296","DOI":"10.1016\/j.trac.2025.118296","article-title":"Engineering CRISPR\/Cas12-based biosensors: Recent advances and future perspectives","volume":"191","author":"Yin","year":"2025","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"287","DOI":"10.18097\/pbmc20247005287","article-title":"Biosensing platforms for DNA diagnostics based on CRISPR\/Cas nucleases: Towards the detection of nucleic acids at the level of single molecules in non-laboratory settings","volume":"70","author":"Khmeleva","year":"2024","journal-title":"Biomed. Khim."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Das, A., Goswami, H.N., Whyms, C.T., Sridhara, S., and Li, H. (2022). Structural principles of CRISPR-Cas enzymes used in nucleic acid detection. J. Struct. Biol., 214.","DOI":"10.1016\/j.jsb.2022.107838"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1465","DOI":"10.1111\/pbi.14051","article-title":"The engineered CRISPR-Mb2Cas12a variant enables sensitive and fast nucleic acid-based pathogens diagnostics in the field","volume":"21","author":"Jiao","year":"2023","journal-title":"Plant Biotechnol. J."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"340938","DOI":"10.1016\/j.aca.2023.340938","article-title":"Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM","volume":"1248","author":"Lin","year":"2023","journal-title":"Anal. Chim. Acta"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Zhu, Z., Li, R., Zhang, H., Wang, J., Lu, Y., Zhang, D., and Yang, L. (2022). PAM-free loop-mediated isothermal amplification coupled with CRISPR\/Cas12a cleavage (Cas-PfLAMP) for rapid detection of rice pathogens. Biosens. Bioelectron., 204.","DOI":"10.1016\/j.bios.2022.114076"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Ptitsyn, K.G., Khmeleva, S.A., Kurbatov, L.K., Timoshenko, O.S., Suprun, E.V., Radko, S.P., and Lisitsa, A.V. (2024). Lamp Primer Designing Software: The Overview. Biomed. Chem. Res. Meth., 7.","DOI":"10.18097\/BMCRM00226"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1006\/mcpr.2002.0415","article-title":"Accelerated reaction by loop-mediated isothermal amplification using loop primers","volume":"16","author":"Nagamine","year":"2002","journal-title":"Mol. Cell. Probes"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Gnanamanickam, S.S. (2006). Clavibacter michiganensis, a group of gram-positive phytopathogenic bacteria. Plant-Associated Bacteria, Springer International Publishing.","DOI":"10.1007\/978-1-4020-4538-7"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1111\/jam.14128","article-title":"Development of a loop-mediated isothermal amplification assay for specific detection of all known subspecies of Clavibacter michiganensis","volume":"126","author":"Dobhal","year":"2019","journal-title":"J. Appl. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Tambong, J.T. (2017). Comparative genomics of Clavibacter michiganensis subspecies, pathogens of important agricultural crops. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0172295"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1006\/bbrc.2001.5921","article-title":"Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation","volume":"289","author":"Mori","year":"2001","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1499","DOI":"10.1042\/BST20190564","article-title":"Making the cut(s): How Cas12a cleaves target and non-target DNA","volume":"47","author":"Swarts","year":"2019","journal-title":"Biochem. Soc. Trans."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"124220","DOI":"10.1016\/j.talanta.2022.124220","article-title":"Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with recombinase polymerase amplification","volume":"255","author":"Hao","year":"2023","journal-title":"Talanta"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Bandyopadhyay, A., Kancharla, N., Javalkote, V.S., Dasgupta, S., and Brutnell, T.P. (2020). CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement. Front. Plant Sci., 11.","DOI":"10.3389\/fpls.2020.584151"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1080\/14737159.2021.1873769","article-title":"LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community","volume":"21","author":"Moehling","year":"2021","journal-title":"Expert Rev. Mol. Diagn."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1016\/j.bj.2019.10.005","article-title":"CRISPR-Cas12a: Functional overview and applications","volume":"43","author":"Paul","year":"2020","journal-title":"Biomed. J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.molcel.2017.03.016","article-title":"Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a","volume":"66","author":"Swarts","year":"2017","journal-title":"Mol. Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1016\/j.molcel.2018.06.043","article-title":"Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a","volume":"71","author":"Strohkendl","year":"2018","journal-title":"Mol. Cell"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Baranova, S.V., Zhdanova, P.V., Pestryakov, P.E., Chernonosov, A.A., and Koval, V.V. (2025). Key thermodynamic characteristics of Cas9 and Cas12a endonucleases\u2019 cleavage of a DNA substrate containing a nucleotide mismatch in the region complementary to RNA. Biochem. Biophys. Res. Commun., 768.","DOI":"10.1016\/j.bbrc.2025.151892"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Mao, X., Xu, J., Jiang, J., Li, Q., Yao, P., Jiang, J., Gong, L., Dong, Y., Tu, B., and Wang, R. (2024). Iterative crRNA design and a PAM-free strategy enabled an ultra-specific RPA-CRISPR\/Cas12a detection platform. Commun. Biol., 7.","DOI":"10.1038\/s42003-024-07173-7"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"4906","DOI":"10.1038\/s41467-020-18615-1","article-title":"Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection","volume":"11","author":"Nguyen","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"5409","DOI":"10.1038\/s41467-023-41006-1","article-title":"Programmable RNA detection with CRISPR-Cas12a","volume":"14","author":"Rananaware","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_31","first-page":"127","article-title":"Limit of Blank (LOB), Limit of Detection (LOD), and Limit of Quantification (LOQ)","volume":"7","author":"Taleuzzaman","year":"2018","journal-title":"Org. Med. Chem. Int. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1023\/A:1008736017029","article-title":"Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by Multiplex PCR with Coamplification of Host DNA","volume":"106","author":"Pastrik","year":"2000","journal-title":"Eur. J. Plant Pathology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1094\/PDIS-93-6-0649","article-title":"A Real-Time PCR Assay for the Detection of Clavibacter michiganensis subsp. sepedonicus Based on the Cellulase A Gene Sequence","volume":"93","author":"Gudmestad","year":"2009","journal-title":"Plant Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"123","DOI":"10.5423\/PPJ.OA.02.2015.0019","article-title":"Validation and Application of a Real-time PCR Protocol for the Specific Detection and Quantification of Clavibacter michiganensis subsp. sepedonicus in Potato","volume":"31","author":"Cho","year":"2015","journal-title":"Plant Pathol. J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"20393","DOI":"10.1038\/s41598-019-56680-9","article-title":"Detection of Potato ring rot Pathogen Clavibacter michiganensis subsp. sepedonicus by Loop-mediated isothermal amplification (LAMP) assay","volume":"9","author":"Sagcan","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"840","DOI":"10.1046\/j.1365-2672.2002.01765.x","article-title":"Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon","volume":"93","author":"Stead","year":"2002","journal-title":"J. Appl. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Khmeleva, S.A., Kurbatov, L.K., Ptitsyn, K.G., Timoshenko, O.S., Morozova, D.D., Suprun, E.V., Radko, S.P., and Lisitsa, A.V. (2024). Detection of Potato Pathogen Clavibacter sepedonicus by CRISPR\/Cas13a Analysis of NASBA Amplicons. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms252212218"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"113458","DOI":"10.1016\/j.microc.2025.113458","article-title":"Loop-mediated isothermal amplification with tyrosine modified 2\u2032-deoxyuridine-5\u2032-triphosphate: Detection of potato pathogen Clavibacter sepedonicus by direct voltammetry of LAMPlicons","volume":"212","author":"Suprun","year":"2025","journal-title":"Microchem. J."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1134\/S0003683824010095","article-title":"Application of DETECTR for Selective Detection of Bacterial Phytopathogen Dickeya solani Using Recombinant CRISPR-Nuclease Cas12a Obtained by Single-Stage Chromatographic Purification","volume":"60","author":"Kurbatov","year":"2024","journal-title":"Appl. Biochem. Microbiol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/16\/8014\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:31:04Z","timestamp":1760034664000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/16\/8014"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,19]]},"references-count":39,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["ijms26168014"],"URL":"https:\/\/doi.org\/10.3390\/ijms26168014","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,19]]}}}