{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,12]],"date-time":"2026-05-12T15:23:26Z","timestamp":1778599406097,"version":"3.51.4"},"reference-count":46,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,11,26]],"date-time":"2024-11-26T00:00:00Z","timestamp":1732579200000},"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. Cell. Infect. Microbiol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p><jats:italic>Salmonella<\/jats:italic>, <jats:italic>Escherichia coli<\/jats:italic>, <jats:italic>Lawsonella intracellularis<\/jats:italic>, and <jats:italic>Brachyspira hyodysenteriae<\/jats:italic> are the primary pathogens responsible for gastrointestinal diseases in pigs, posing a significant threat to the health and productivity of pig production systems. Pathogen detection is a crucial tool for monitoring and managing these infections.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We designed primers and probes targeting the <jats:italic>invA<\/jats:italic> gene of <jats:italic>Salmonella<\/jats:italic>, the 23S <jats:italic>rRNA<\/jats:italic> gene of <jats:italic>Escherichia coli<\/jats:italic>, the <jats:italic>aspA<\/jats:italic> gene of <jats:italic>Lawsonella intracellularis<\/jats:italic>, and the <jats:italic>nox<\/jats:italic> gene of <jats:italic>Brachyspira hyodysenteriae<\/jats:italic>. We developed a quadruplex TaqMan real-time quantitative PCR assay capable of simultaneously detecting these four pathogens.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>This assay demonstrated high sensitivity, with detection limits of 100 copies\/\u03bcL for the recombinant plasmid standards pEASY-23S rRNA, pEASY-aspA, and pEASY-<jats:italic>nox<\/jats:italic>, and 10 copies\/\u03bcL for pEASY-<jats:italic>invA<\/jats:italic>. The standard curves exhibited excellent linearity (R<jats:sup>2<\/jats:sup> values of 0.999, 0.999, 1, and 0.998, respectively) and high amplification efficiencies (93.57%, 94.84%, 85.15%, and 81.81%, respectively). The assay showed high specificity, with no cross-reactivity detected against nucleic acids from Streptococcus suis, porcine epidemic diarrhoea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), <jats:italic>Pasteurella multocida<\/jats:italic>, <jats:italic>Clostridium perfringens<\/jats:italic>, <jats:italic>Gracilaria parapsilosis<\/jats:italic>, porcine delta coronavirus (PDCoV), porcine group A rotavirus (GARV), and porcine teschovirus (PTV). The assay also exhibited excellent repeatability, with inter- and intra-assay coefficient of variation (CV) ranging from 0.15% to 1.12%. High concentrations of nucleic acids did not interfere with the detection of low concentrations, ensuring robust performance in complex samples. Among 263 diarrhoeic samples, the assay detected <jats:italic>Salmonella<\/jats:italic> in 23.95%, <jats:italic>Escherichia coli<\/jats:italic> in 26.24%, <jats:italic>Lawsonella intracellularis<\/jats:italic> in 33.84%, and <jats:italic>Brachyspira hyodysenteriae<\/jats:italic> in 22.43%.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>This quadruplex TaqMan qPCR assay offers a rapid, sensitive, and specific tool for the simultaneous detection of <jats:italic>Salmonella<\/jats:italic>, <jats:italic>Escherichia coli<\/jats:italic>, <jats:italic>Lawsonella intracellularis<\/jats:italic>, and <jats:italic>Brachyspira hyodysenteriae<\/jats:italic> in pigs.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fcimb.2024.1468783","type":"journal-article","created":{"date-parts":[[2024,11,26]],"date-time":"2024-11-26T16:18:15Z","timestamp":1732637895000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":5,"title":["Development and application of a quadruplex TaqMan real-time fluorescence quantitative PCR assay for four porcine digestive pathogens"],"prefix":"10.3389","volume":"14","author":[{"given":"Haojie","family":"Wang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Sun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianxing","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Haibo","family":"Yu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caixia","family":"Gao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tongqing","family":"An","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongyan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liangquan","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhimin","family":"Jin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Changqing","family":"Yu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Changyou","family":"Xia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"He","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2024,11,26]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"105862","DOI":"10.1016\/j.prevetmed.2023.105862","article-title":"Prevalence and risk factors of Brachyspira spp. in pig herds with a history of diarrhoea in six European countries","volume":"213","author":"Arnold","year":"2023","journal-title":"Prev. 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