{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:11:12Z","timestamp":1785409872066,"version":"3.56.0"},"reference-count":40,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"China Agriculture Research System of MOF and MARA","award":["CARS-29"],"award-info":[{"award-number":["CARS-29"]}]},{"award":["CARS-29"],"award-info":[{"award-number":["CARS-29"]}],"id":[{"id":"https:\/\/ror.org\/03p5eky76","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100007934","name":"Outstanding Scientist Project of Beijing Academy of Agriculture and Forestry Sciences","doi-asserted-by":"publisher","award":["JKTD2025002"],"award-info":[{"award-number":["JKTD2025002"]}],"id":[{"id":"10.13039\/501100007934","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>Grapevine black-foot disease is a destructive trunk disease with a complex pathogen composition that often involves mixed and latent infections, making timely field diagnosis challenging. To improve rapid field detection, we developed a rapid, sensitive, and low instrument-dependent nucleic acid assay. The assay integrates recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)\u2013Cas12a for the detection of Ilyonectria and Dactylonectria, two genera associated with grapevine black-foot disease. Conserved regions of the histone H3 and \u03b2-tubulin genes were selected for the design of specific RPA primers and corresponding CRISPR RNAs (crRNAs) for Ilyonectria and Dactylonectria, respectively. A workflow integrating RPA, Cas12a-mediated recognition, and lateral flow assay (LFA)-based visualization was established. The reaction conditions were optimized to enhance amplification efficiency and Cas12a recognition stability. Specificity was evaluated using DNA from target and non-target fungi, and sensitivity was determined using serially diluted templates. Under optimized conditions, the assay detected Ilyonectria DNA at concentrations as low as 3.6 ng\/\u03bcL within 1 h at 39 \u00b0C. For Dactylonectria, the detection limit reached 80 fg\/\u03bcL within 50 min at 41 \u00b0C. No cross-reactivity was observed. The LFA strips exhibited positive and negative bands within minutes, enabling rapid visual interpretation. This RPA-CRISPR\/Cas12a-LFA system provides a rapid, visually interpretable approach for detecting selected grapevine black-foot disease-associated species in China. The workflow reduces the requirement for specialized thermocycling and fluorescence detection equipment during amplification and readout, following DNA extraction.<\/jats:p>","DOI":"10.3390\/jof12070455","type":"journal-article","created":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T01:03:23Z","timestamp":1782263003000},"page":"455","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Rapid Recombinase Polymerase Amplification\u2013CRISPR\/Cas12a Assay for Detecting Grapevine Black-Foot Pathogens"],"prefix":"10.3390","volume":"12","author":[{"given":"Wenwen","family":"Liang","sequence":"first","affiliation":[{"name":"College of Plant Protection, Hebei Agricultural University, 2596 Lekai South Street, Lianchi District, Baoding 071000, China"},{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2550-7089","authenticated-orcid":false,"given":"Baoyu","family":"Wang","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1872-0968","authenticated-orcid":false,"given":"Junbo","family":"Peng","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1359-0412","authenticated-orcid":false,"given":"Caiping","family":"Huang","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yueyan","family":"Zhou","sequence":"additional","affiliation":[{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xing","family":"Li","sequence":"additional","affiliation":[{"name":"College of Plant Protection, Hebei Agricultural University, 2596 Lekai South Street, Lianchi District, Baoding 071000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6814-5412","authenticated-orcid":false,"given":"Wei","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Plant Protection, Hebei Agricultural University, 2596 Lekai South Street, Lianchi District, Baoding 071000, China"},{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiye","family":"Yan","sequence":"additional","affiliation":[{"name":"College of Plant Protection, Hebei Agricultural University, 2596 Lekai South Street, Lianchi District, Baoding 071000, China"},{"name":"Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,23]]},"reference":[{"key":"ref_1","first-page":"278","article-title":"Occurrence of Grapevine Trunk Diseases Caused by Fungal Pathogens in the Domestic and Overseas","volume":"38","author":"Ye","year":"2021","journal-title":"J. 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