{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T05:16:59Z","timestamp":1787894219598,"version":"build-2784847793"},"reference-count":28,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Science and Technology Projects of Xizang Autonomous Region","award":["XZ202501ZY0122"],"award-info":[{"award-number":["XZ202501ZY0122"]}]},{"name":"Science and Technology Projects of Xizang Autonomous Region","award":["XZ202401JD0012"],"award-info":[{"award-number":["XZ202401JD0012"]}]},{"DOI":"10.13039\/501100021171","name":"Guangdong Basic and Applied Basic Research Foundation","doi-asserted-by":"crossref","award":["2021B1515120006"],"award-info":[{"award-number":["2021B1515120006"]}],"id":[{"id":"10.13039\/501100021171","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Curr. Issues Mol. Biol.","CIMB"],"abstract":"<jats:p>The strict requirement for a canonical TTTV protospacer adjacent motif (PAM) restricts target-site selection in clustered regularly interspaced short palindromic repeats\/CRISPR-associated protein 12a (CRISPR\/Cas12a)-based diagnostics and limits the flexibility of assay design. Whether suboptimal PAMs can be effectively incorporated into one-pot detection systems for Clostridium perfringens has not been systematically examined. Here, we developed two one-pot recombinase polymerase amplification (RPA)\u2013CRISPR\/Cas12a assays targeting the plc and cpe toxin genes of C. perfringens and systematically evaluated 28 crRNAs spanning canonical (TTTV) and suboptimal (VTTV, TCTV) PAMs in both trans-cleavage and one-pot formats. Several suboptimal-PAM crRNAs, including crRNA 4 for plc (PAM: GTTG) and crRNA 7 for cpe (PAM: CTTA), maintained efficient detection in the one-pot system, whereas crRNA performance between the two formats was not predictable, underscoring the need for direct one-pot screening. The optimized assays achieved limits of detection of 50 copies (plc) and 10 copies (cpe), showed no cross-reactivity against four non-target bacterial species. The plc assay was successfully validated in pork, chicken, and beef samples spiked with C. perfringens, with results readable within 40 min using a simple isothermal device and naked-eye readout. The cpe assay was confirmed as a proof-of-concept at the genomic DNA level. These results demonstrate that suboptimal PAMs can serve as a deliberate design strategy to expand target accessibility in one-pot CRISPR\/Cas12a diagnostics.<\/jats:p>","DOI":"10.3390\/cimb48090867","type":"journal-article","created":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T16:54:42Z","timestamp":1787763282000},"page":"867","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Suboptimal PAM Enables One-Pot RPA\u2013CRISPR\/Cas12a Detection of Clostridium perfringens in Food Samples"],"prefix":"10.3390","volume":"48","author":[{"given":"Bo","family":"Li","sequence":"first","affiliation":[{"name":"School of Animal Science and Technology, Foshan University, Foshan 528225, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6423-3060","authenticated-orcid":false,"given":"Xin","family":"Li","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kai","family":"Li","sequence":"additional","affiliation":[{"name":"School of Animal Science and Technology, Foshan University, Foshan 528225, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shenquan","family":"Liao","sequence":"additional","affiliation":[{"name":"Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingfei","family":"Sun","sequence":"additional","affiliation":[{"name":"Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3613-2350","authenticated-orcid":false,"given":"Fuqiang","family":"Huang","sequence":"additional","affiliation":[{"name":"School of Animal Science and Technology, Foshan University, Foshan 528225, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haoji","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Animal Science and Technology, Foshan University, Foshan 528225, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1089\/fpd.2012.1316","article-title":"Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, united states, 1998\u20132010","volume":"10","author":"Grass","year":"2013","journal-title":"Foodborne Pathog. 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