{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:56:34Z","timestamp":1787028994798,"version":"build-2736575974"},"reference-count":32,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,14]],"date-time":"2025-08-14T00:00:00Z","timestamp":1755129600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Nature Science Foundation of China","award":["82372283"],"award-info":[{"award-number":["82372283"]}]},{"name":"National Nature Science Foundation of China","award":["Ym2023085"],"award-info":[{"award-number":["Ym2023085"]}]},{"name":"Jiangsu Province Preventive Medicine Research Project","award":["82372283"],"award-info":[{"award-number":["82372283"]}]},{"name":"Jiangsu Province Preventive Medicine Research Project","award":["Ym2023085"],"award-info":[{"award-number":["Ym2023085"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Foodborne pathogens represent a class of pathogenic microorganisms capable of causing food poisoning or serving as foodborne vectors, constituting a major source of food safety concerns. With increasing demands for rapid diagnostics, conventional culture-based methods and PCR assays face limitations due to prolonged turnaround times and specialized facility requirements. While CRISPR-based detection has emerged as a promising rapid diagnostic platform, its inherent inability to detect low-abundance targets necessitates coupling with isothermal amplification, thereby increasing operational complexity. In this study, we preliminarily developed a novel amplification-free Cascade-CRISPR detection system utilizing a hairpin DNA amplifier. This method achieves detection sensitivity as low as 10 fM (82 parasites\/\u03bcL) for DNA targets within 30 min without requiring pre-amplification, with background signal suppression achieved through optimized NaCl concentration. Validation using artificially contaminated food samples demonstrated the platform\u2019s robust performance for both Toxoplasma gondii (T. gondii) and Listeria monocytogenes (L. monocytogenes) detection, confirming broad applicability. In summary, this study preliminarily establishes an amplification-free Cascade-CRISPR detection platform that achieves high sensitivity and rapid turnaround, demonstrating strong potential for on-site screening of foodborne pathogens.<\/jats:p>","DOI":"10.3390\/microorganisms13081896","type":"journal-article","created":{"date-parts":[[2025,8,14]],"date-time":"2025-08-14T14:51:46Z","timestamp":1755183106000},"page":"1896","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Cascade CRISPR\/cas Enables More Sensitive Detection of Toxoplasma gondii and Listeria monocytogenes than Single CRISPR\/cas"],"prefix":"10.3390","volume":"13","author":[{"given":"Dawei","family":"Chen","sequence":"first","affiliation":[{"name":"NHC Key Laboratory on Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Chinese Center for Tropical Diseases Research, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, WHO Collaborating Center for Tropical Diseases, Shanghai 200025, China"},{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Sun","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory on Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Chinese Center for Tropical Diseases Research, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, WHO Collaborating Center for Tropical Diseases, Shanghai 200025, China"},{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingbing","family":"Li","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Ma","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qinjun","family":"Zhang","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanli","family":"Yin","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Li","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingyue","family":"Wei","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Liu","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengfei","family":"Yang","sequence":"additional","affiliation":[{"name":"Huai\u2019an Center for Disease Control and Prevention, Jiangsu Provincial Regional Public Health Center, Huai\u2019an 223003, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4386-6624","authenticated-orcid":false,"given":"Yujuan","family":"Shen","sequence":"additional","affiliation":[{"name":"NHC Key Laboratory on Parasite and Vector Biology, National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Chinese Center for Tropical Diseases Research, National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, WHO Collaborating Center for Tropical Diseases, Shanghai 200025, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,14]]},"reference":[{"key":"ref_1","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_2","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1038\/s41421-018-0028-z","article-title":"CRISPR-Cas12a-assisted nucleic acid detection","volume":"4","author":"Li","year":"2018","journal-title":"Cell Discov."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1126\/science.aav4294","article-title":"Programmed DNA destruction by miniature CRISPR-Cas14 enzymes","volume":"362","author":"Harrington","year":"2018","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1038\/s41587-024-02255-7","article-title":"Trans-nuclease activity of Cas9 activated by DNA or RNA target binding","volume":"43","author":"Chen","year":"2025","journal-title":"Nat. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"685","DOI":"10.2147\/IDR.S348456","article-title":"A New Method Based on LAMP-CRISPR-Cas12a-Lateral Flow Immunochromatographic Strip for Detection","volume":"15","author":"Xu","year":"2022","journal-title":"Infect. Drug Resist."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Chen, H., Zhang, H., Guo, J., Meng, X., Yao, M., He, L., Nie, X., Xu, H., Liu, C., and Sun, J. (2025). Rapid detection of feline parvovirus using RAA-CRISPR\/Cas12a-based lateral flow strip and fluorescence. Front. Microbiol., 16.","DOI":"10.3389\/fmicb.2025.1501635"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Wang, J., Zhu, X., Yin, D., Cai, C., Liu, H., Yang, Y., Guo, Z., Yin, L., Shen, X., and Dai, Y. (2023). Rapid and Easy-Read Porcine Circovirus Type 4 Detection with CRISPR-Cas13a-Based Lateral Flow Strip. Microorganisms, 11.","DOI":"10.3390\/microorganisms11020354"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Shen, J., Chen, Z., Xie, R., Li, J., Liu, C., He, Y., Ma, X., Yang, H., and Xie, Z. (2023). CRISPR\/Cas12a-Assisted isothermal amplification for rapid and specific diagnosis of respiratory virus on an microfluidic platform. Biosens. Bioelectron., 237.","DOI":"10.1016\/j.bios.2023.115523"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3463","DOI":"10.1094\/PDIS-06-24-1300-SR","article-title":"RPA-CRISPR\/Cas12a-Mediated Isothermal Amplification for Rapid Detection of Phytopythium helicoides","volume":"108","author":"Xu","year":"2024","journal-title":"Plant Dis."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Xu, D., Zeng, H., Wu, W., Liu, H., and Wang, J. (2023). Isothermal Amplification and CRISPR\/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus. Foods, 12.","DOI":"10.3390\/foods12244432"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1038\/s41551-022-00861-x","article-title":"Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a","volume":"6","author":"Lu","year":"2022","journal-title":"Nat. Biomed. Eng."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR-Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4711","DOI":"10.1038\/s41467-020-18575-6","article-title":"Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay","volume":"11","author":"Ding","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1410","DOI":"10.1016\/j.tibtech.2024.06.009","article-title":"One-pot diagnostic methods based on CRISPR\/Cas and Argonaute nucleases: Strategies and perspectives","volume":"42","author":"Ye","year":"2024","journal-title":"Trends Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1039\/D0CC06412B","article-title":"Cascade CRISPR\/cas enables amplification-free microRNA sensing with fM-sensitivity and single-base-specificity","volume":"57","author":"Sha","year":"2021","journal-title":"Chem. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"eabc7802","DOI":"10.1126\/sciadv.abc7802","article-title":"A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics","volume":"7","author":"Shi","year":"2021","journal-title":"Sci. Adv."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e2420166122","DOI":"10.1073\/pnas.2420166122","article-title":"Amplification-free, OR-gated CRISPR-Cascade reaction for pathogen detection in blood samples","volume":"122","author":"Lim","year":"2025","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1586\/14787210.2016.1146131","article-title":"Role of vertical transmission of Toxoplasma gondii in prevalence of infection","volume":"14","author":"Hide","year":"2016","journal-title":"Expert. Rev. Anti Infect. Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S1043-4526(10)60001-0","article-title":"Transmission of toxoplasmosis (Toxoplasma gondii) by foods","volume":"60","author":"Pereira","year":"2010","journal-title":"Adv. Food Nutr. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1080\/20477724.2022.2083977","article-title":"Cerebral toxoplasmosis in HIV-infected patients: A review","volume":"117","author":"Dian","year":"2023","journal-title":"Pathog. Glob. Health"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1128\/mr.55.3.476-511.1991","article-title":"Listeria monocytogenes, a food-borne pathogen","volume":"55","author":"Farber","year":"1991","journal-title":"Microbiol. Rev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"529","DOI":"10.3934\/microbiol.2017.3.529","article-title":"Foodborne pathogens","volume":"3","author":"Bintsis","year":"2017","journal-title":"AIMS Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Jeung, J.H., Han, H., Lee, C.Y., and Ahn, J.K. (2023). CRISPR\/Cas12a Collateral Cleavage Activity for Sensitive 3\u2032-5\u2032 Exonuclease Assay. Biosensors, 13.","DOI":"10.3390\/bios13110963"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2228","DOI":"10.1021\/acssynbio.9b00209","article-title":"HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation","volume":"8","author":"Li","year":"2019","journal-title":"ACS Synth. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Aladhadh, M. (2023). A Review of Modern Methods for the Detection of Foodborne Pathogens. Microorganisms, 11.","DOI":"10.3390\/microorganisms11051111"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Bai, W., Chen, J., Chen, D., Zhu, Y., Hu, K., Lin, X., Chen, J., and Song, D. (2024). Sensitive and rapid detection of three foodborne pathogens in meat by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD). Int. J. Food Microbiol., 422.","DOI":"10.1016\/j.ijfoodmicro.2024.110822"},{"key":"ref_27","first-page":"100183","article-title":"Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities","volume":"7","author":"Ndraha","year":"2023","journal-title":"Food Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1007\/s10068-022-01160-6","article-title":"Review in isothermal amplification technology in food microbiological detection","volume":"31","author":"Li","year":"2022","journal-title":"Food Sci. Biotechnol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1818","DOI":"10.1038\/s41467-024-46001-8","article-title":"Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA\/RNA sensing","volume":"15","author":"Deng","year":"2024","journal-title":"Nat. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e39","DOI":"10.1093\/nar\/gkae176","article-title":"An autocatalytic CRISPR-Cas amplification effect propelled by the LNA-modified split activators for DNA sensing","volume":"52","author":"Sun","year":"2024","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"28866","DOI":"10.1021\/acsomega.4c03413","article-title":"Rapid and Amplification-free Nucleic Acid Detection with DNA Substrate-Mediated Autocatalysis of CRISPR\/Cas12a","volume":"9","author":"Zhou","year":"2024","journal-title":"ACS Omega"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Hwang, I., Song, Y.H., and Lee, S. (2025). Enhanced trans-cleavage activity using CRISPR-Cas12a variant designed to reduce steric inhibition by cis-cleavage products. Biosens. Bioelectron., 267.","DOI":"10.1016\/j.bios.2024.116859"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/8\/1896\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:27:35Z","timestamp":1760034455000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/8\/1896"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,14]]},"references-count":32,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["microorganisms13081896"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms13081896","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,14]]}}}