{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T14:45:55Z","timestamp":1785854755814,"version":"3.56.0"},"reference-count":52,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"abstract":"<jats:p>\n                    Tobacco production is increasingly threatened by pathogens such as\n                    <jats:italic>Ralstonia<\/jats:italic>\n                    ,\n                    <jats:italic>Phytophthora<\/jats:italic>\n                    ,\n                    <jats:italic>Alternaria<\/jats:italic>\n                    , and\n                    <jats:italic>Pseudomonas<\/jats:italic>\n                    , which cause bacterial wilt, black shank, brown spot, and wildfire, respectively. Conventional diagnostics typically target single pathogens, and reliable methods for simultaneous multi-pathogen detection remain limited. Here, we developed a multiplex PCR\u2013Cas12a\u2013LFD platform for the pooled multi-target screening of four major tobacco pathogens. Multiplex PCR amplifies pathogen-specific 16S rRNA and ITS regions, while Cas12a\u2013crRNA complexes specifically recognize the amplicons and trigger trans-cleavage of fluorophore\u2013quencher or fluorophore\u2013biotin probes, enabling fluorescence and lateral flow readouts. Reaction parameters were systematically optimized to reduce primer interference and achieve balanced amplification across targets. The individual target groups showed different mass-based analytical LODs, ranging from 5 fg\/\u03bcL to 5 pg\/\u03bcL under the tested conditions. For an equal-proportion mixed-template series, the universal reporter produced a pooled-signal LOD of 186 fg\/\u03bcL and showed no detectable signal from the tested non-target DNA panel. LFD-based visual detection reached 0.5 pg\/\u03bcL, while preliminary spiked-leaf tests at two extracted-DNA levels yielded apparent recovery rates of 93%\u201395%, indicating matrix compatibility under the tested conditions. This platform provides a laboratory-based pooled screening tool for preliminary sample triage, with target-specific identification requiring subsequent confirmatory analysis.\n                  <\/jats:p>","DOI":"10.3389\/fpls.2026.1919480","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T13:46:36Z","timestamp":1785851196000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["A multiplex PCR\u2013Cas12a\u2013lateral flow platform for pooled screening of four major tobacco pathogen groups"],"prefix":"10.3389","volume":"17","author":[{"given":"Yena","family":"Zhang","sequence":"first","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]},{"name":"College of Plant Protection, Southwest University","place":["Chongqing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changwen","family":"Ye","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Zeng","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongru","family":"Xi","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baojian","family":"Wu","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kuo","family":"Huang","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dandan","family":"Liu","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guokang","family":"Chen","sequence":"additional","affiliation":[{"name":"College of Plant Protection, Southwest University","place":["Chongqing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chenqi","family":"Niu","sequence":"additional","affiliation":[{"name":"China Tobacco Standardization Research Center, Zhengzhou Tobacco Research Institute of CNTC","place":["Zhengzhou, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,4]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.rhisph.2022.100479","article-title":"Ralstonia solanacearum, a deadly pathogen: Revisiting the bacterial wilt biocontrol practices in tobacco and other Solanaceae","volume":"21","author":"Ahmed","year":"2022","journal-title":"Rhizosphere"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/j.heliyon.2023.e14505","article-title":"Environmental impact assessment of tobacco farming in northern Bangladesh","volume":"9","author":"Ali","year":"2023","journal-title":"Heliyon"},{"key":"B3","doi-asserted-by":"publisher","first-page":"1226","DOI":"10.1021\/acssynbio.9b00507","article-title":"Nucleic acid detection using CRISPR\/Cas biosensing technologies","volume":"9","author":"Aman","year":"2020","journal-title":"ACS Synth. 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