{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T22:29:22Z","timestamp":1785364162173,"version":"3.55.0"},"reference-count":41,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T00:00:00Z","timestamp":1751241600000},"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. Microbiol."],"abstract":"<jats:sec><jats:title>Background<\/jats:title><jats:p>The quail farming industry constitutes an important component of China\u2019s agricultural sector. However, it is frequently threatened by various bacterial and mycoplasmal infections, particularly respiratory diseases caused by <jats:italic>Pasteurella multocida<\/jats:italic>, <jats:italic>Avibacterium paragallinarum<\/jats:italic>, <jats:italic>Mycoplasma gallisepticum<\/jats:italic>, and <jats:italic>Mycoplasma synoviae<\/jats:italic>. These pathogens commonly result in co-infections or secondary infections, and their clinical presentations are often indistinguishable due to the similarity of symptoms.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Four sets of primers and probes were designed based on the GenBank-registered gene sequences: the <jats:italic>kmt1<\/jats:italic> gene of <jats:italic>P. multocida<\/jats:italic>, the <jats:italic>recN<\/jats:italic> gene of <jats:italic>A. paragallinarum<\/jats:italic>, the <jats:italic>mgc2<\/jats:italic> gene of <jats:italic>M. gallisepticum<\/jats:italic>, and the <jats:italic>vlhA<\/jats:italic> gene of <jats:italic>M. synoviae<\/jats:italic>. Reaction conditions were optimized accordingly. A recombinant plasmid standard was constructed for the generation of standard curves. The sensitivity, specificity, reproducibility, and accuracy of the assay were systematically evaluated.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The constructed standard curves demonstrated strong linearity (<jats:italic>R<\/jats:italic><jats:sup>2<\/jats:sup>\u202f=\u202f1.000, 0.998, 1.000, and 1.000), with high amplification efficiencies (107.09, 91.23, 112.10, and 125.51%, respectively). The detection limit for each recombinant plasmid standard was as low as 10 copies. No cross-reactivity was observed with non-target pathogens, including avian pox virus, <jats:italic>Escherichia coli<\/jats:italic>, <jats:italic>Salmonella<\/jats:italic> spp., Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and <jats:italic>Staphylococcus aureus<\/jats:italic>. The assay exhibited excellent reproducibility, with inter- and intra-assay coefficient of variation (CV) values ranging from 0.11 to 1.41%. Among 126 clinical samples, <jats:italic>P. multocida<\/jats:italic> was detected in 6 samples, <jats:italic>A. paragallinarum<\/jats:italic> in 3, <jats:italic>M. gallisepticum<\/jats:italic> in 6, and <jats:italic>M. synoviae<\/jats:italic> in 4. These results were consistent with those obtained using previously established methods.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>A highly sensitive, specific, rapid, and efficient quadruplex fluorescence quantitative PCR assay was successfully developed for the simultaneous detection and identification of <jats:italic>Pasteurella multocida<\/jats:italic>, <jats:italic>Avibacterium paragallinarum<\/jats:italic>, <jats:italic>Mycoplasma gallisepticum<\/jats:italic>, and <jats:italic>Mycoplasma synoviae<\/jats:italic>.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fmicb.2025.1605356","type":"journal-article","created":{"date-parts":[[2025,6,30]],"date-time":"2025-06-30T05:26:25Z","timestamp":1751261185000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["The quadruplex fluorescent quantitative PCR method for the simultaneous detection of respiratory diseases in quail: Pasteurella multocida, Avibacterium paragallinarum, Mycoplasma gallisepticum, and Mycoplasma synoviae"],"prefix":"10.3389","volume":"16","author":[{"given":"Haojie","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lihong","family":"Xue","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Longxi","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yixuan","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianxing","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tongqing","family":"An","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changwen","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changqing","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Changyou","family":"Xia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"He","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,6,30]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"1803","DOI":"10.1002\/vms3.1166","article-title":"Isolation and identification of Mannheimia haemolytica and Pasteurella multocida from symptomatic and asymptomatic sheep and their antibiotic susceptibility patterns in three selected districts of North Gondar zone, Gondar Ethiopia","volume":"9","author":"Abate","year":"2023","journal-title":"Vet. 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