{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T18:09:59Z","timestamp":1778868599913,"version":"3.51.4"},"reference-count":29,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T00:00:00Z","timestamp":1778803200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Neonatal calf diarrhea, primarily caused by bovine viral diarrhea virus (BVDV) and enterotoxigenic\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    K99, poses a significant threat to the cattle industry. This study aimed to develop a multiplex qPCR assay for the simultaneous detection of both pathogens. We established specific primers and probes and generated optimized qPCR standard curves, achieving high analytical sensitivity, with limits of detection of 10\u00b2 copies\/\u03bcL for the BVDV target fragment and 10\u00b9 copies\/\u03bcL for the\n                    <jats:italic>E. coli<\/jats:italic>\n                    K99 plasmid DNA, without cross-reactivity with other bovine pathogens. The assay demonstrated good repeatability and was validated using clinical samples, confirming its effectiveness for accurate diagnostics. This multiplex qPCR assay enhances monitoring of BVDV and\n                    <jats:italic>E. coli<\/jats:italic>\n                    infections in livestock, potentially reducing economic losses from outbreaks.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0349315","type":"journal-article","created":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T17:29:01Z","timestamp":1778866141000},"page":"e0349315","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a multiplex fluorescent qPCR assay for the simultaneous detection of bovine viral diarrhea virus and pathogenic Escherichia coli"],"prefix":"10.1371","volume":"21","author":[{"given":"Wenxin","family":"Zhu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5066-6654","authenticated-orcid":true,"given":"Huanxing","family":"Zhu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2026,5,15]]},"reference":[{"issue":"1","key":"pone.0349315.ref001","doi-asserted-by":"crossref","first-page":"1","DOI":"10.4142\/jvs.2014.15.1.1","article-title":"An overview of calf diarrhea - infectious etiology, diagnosis, and intervention","volume":"15","author":"Y-I Cho","year":"2014","journal-title":"J Vet Sci"},{"key":"pone.0349315.ref002","doi-asserted-by":"crossref","first-page":"3075429","DOI":"10.1155\/2020\/3075429","article-title":"Assessment of Major Causes of Calf Mortality in Urban and Periurban Dairy Production System of 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