{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:29:12Z","timestamp":1787027352004,"version":"build-2736575974"},"reference-count":16,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T00:00:00Z","timestamp":1771804800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Independent Scientific Research Project of Science and Technology Research Center of China Customs","award":["2024HX11"],"award-info":[{"award-number":["2024HX11"]}]},{"name":"Scientific Research Project of the General Administration of Customs","award":["2025HK197"],"award-info":[{"award-number":["2025HK197"]}]},{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2022YFC2602400"],"award-info":[{"award-number":["2022YFC2602400"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:p>\n                    Co-infections involving\n                    <jats:italic>Actinobacillus pleuropneumoniae<\/jats:italic>\n                    (APP) and influenza A (H1N1) virus present a serious diagnostic challenge in swine respiratory disease, complicating effective outbreak management and control. This study reports the development and validation of a novel duplex TaqMan real-time PCR assay, optimized for the Coyote Flash10 portable automated detection system, for the simultaneous identification and quantification of both pathogens. The assay employs primers and probes specific to the apxIVA virulence gene of APP and the conserved matrix (M) gene of H1N1, with porcine RNase P as an internal control. Validation using recombinant plasmid standards demonstrated a sensitivity of 1 copy\/\u03bcL for each target, with strong linear correlation (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u202f&amp;gt;\u202f0.99). Calculated amplification efficiencies (APP: 121.6%; H1N1: 118.6%) were marginally above the typical 90\u2013110% range. However, the assay exhibited robust and consistent quantification without evidence of reaction competition. In a simulated clinical matrix, detection limits corresponded to 10,000-fold and 100-fold dilutions for APP and H1N1, respectively. The method showed excellent repeatability (intra-assay CV &amp;lt;3%) and high specificity, with no cross-reactivity against six other common porcine respiratory pathogens. This rapid, closed-tube assay provides a complete result within one hour and offers a practical, point-of-care-compatible solution for on-farm surveillance and the differential diagnosis of complex respiratory co-infections in swine populations. A key limitation of this study is that validation was performed primarily using simulated samples; future validation with authentic clinical samples will further confirm the method\u2019s clinical utility.\n                  <\/jats:p>","DOI":"10.3389\/fvets.2026.1774637","type":"journal-article","created":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T05:28:18Z","timestamp":1771824498000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["A duplex real-time fluorescent quantitative PCR method for simultaneous and rapid detection of Actinobacillus pleuropneumoniae and influenza A virus"],"prefix":"10.3389","volume":"13","author":[{"given":"Yu-Jie","family":"Ren","sequence":"first","affiliation":[{"name":"Science and Technology Research Center of China Customs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Lin","sequence":"additional","affiliation":[{"name":"Chengdu Customs Technical Center","place":["Chengdu, Sichuan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dao-Jian","family":"Yu","sequence":"additional","affiliation":[{"name":"Animal & Plant Inspection and Quarantine Technology Center of Shenzhen Customs","place":["Shenzhen, Guangdong, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Chengdu Customs Technical Center","place":["Chengdu, Sichuan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Yang","sequence":"additional","affiliation":[{"name":"Science and Technology Research Center of China Customs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bai-Yi","family":"Zhang","sequence":"additional","affiliation":[{"name":"Science and Technology Research Center of China Customs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying-Qi","family":"Chen","sequence":"additional","affiliation":[{"name":"Chengdu Customs Technical Center","place":["Chengdu, Sichuan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hai-Xin","family":"Long","sequence":"additional","affiliation":[{"name":"Beijing Coyote Biotech Co., Ltd.","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li-Li","family":"Ren","sequence":"additional","affiliation":[{"name":"Science and Technology Research Center of China Customs","place":["Beijing, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,2,23]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"3240","DOI":"10.1093\/nar\/gkad091","article-title":"Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions","volume":"51","author":"Nahar","year":"2023","journal-title":"Nucleic Acids Res"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"2387449","DOI":"10.1080\/22221751.2024.2387449","article-title":"Gene editing of pigs to control influenza A virus infections","volume":"13","author":"Kwon","year":"2024","journal-title":"Emerg Microbes Infect"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"128043","DOI":"10.1016\/j.micres.2024.128043","article-title":"The Actinobacillus pleuropneumoniae apxIV operon encodes an antibacterial toxin-immunity 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MDCK suspension cells","volume":"259","author":"Kuchler","year":"2022","journal-title":"J Proteome"},{"key":"ref15","doi-asserted-by":"publisher","first-page":"1040056","DOI":"10.3389\/fmicb.2022.1040056","article-title":"Exploring the active ingredients and pharmacological mechanisms of the oral intake formula Huoxiang Suling Shuanghua decoction on influenza virus type A based on network pharmacology and experimental exploration","volume":"13","author":"Tang","year":"2022","journal-title":"Front Microbiol"},{"key":"ref16","volume-title":"WOAH terrestrial manual 2023","author":"Brown","year":"2023"}],"container-title":["Frontiers in Veterinary 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