{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T19:54:45Z","timestamp":1782849285631,"version":"3.54.5"},"reference-count":34,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":180,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,1,1]],"date-time":"2026-01-01T00:00:00Z","timestamp":1767225600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Transboundary and Emerging Diseases"],"published-print":{"date-parts":[[2026,1]]},"abstract":"<jats:p>\n                    Six key viral pathogens\u2014porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine deltacoronavirus (PDCoV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV)\u2014inflict substantial economic losses on the global swine industry. Their frequent cocirculation and the resulting mixed infections severely complicate rapid and accurate diagnosis. To overcome this challenge, we developed a multiplex real\u2010time PCR assay integrated with high\u2010resolution melting (HRM) analysis for their simultaneous detection and differentiation. Specific primer sets were designed and optimized to target conserved regions of the PEDV\u2010M, TGEV\u2010N, PoRV\u2010VP6, PDCoV\u2010M, PRV\u2010gE, and PRRSV\u2010ORF5 genes. Reaction conditions and HRM parameters were refined, ensuring specific amplification and distinct melting profiles for all six viruses. The assay exhibited excellent specificity, showing no cross\u2010reactivity with other common porcine pathogens. Limits of detection (LODs) were established at 10\u2009copies\/\u03bcL for five viruses (PEDV, TGEV, PoRV, PDCoV, and PRRSV) and 100\u2009copies\/\u03bcL for PRV. Amplification efficiency ranged from 90% to 102% (\n                    <jats:italic>R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u2009\u2265\u20090.98). High reproducibility was confirmed by intra\u2010 and interassay coefficients of variation (CVs) of &lt;3% for cycle threshold (\n                    <jats:italic>C<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>t<\/jats:italic>\n                    <\/jats:sub>\n                    ) values and &lt;0.5% for melting temperature (\n                    <jats:italic>T<\/jats:italic>\n                    <jats:sub>\n                      <jats:italic>m<\/jats:italic>\n                    <\/jats:sub>\n                    ) values. Clinical validation of 248 field samples demonstrated that detection rates for the six pathogens were 16.0%\u221227.3% higher (an average of 18.1%) than those of conventional PCR assays. Collectively, these findings confirm that the HRM\u2010based multiplex real\u2010time PCR assay is a rapid, sensitive, specific, and highly reproducible tool for detecting and differentiating major porcine viral pathogens. This methodology offers a valuable molecular diagnostic platform, supporting early diagnosis, epidemiological surveillance, and enhanced control of swine viral diseases.\n                  <\/jats:p>","DOI":"10.1155\/tbed\/1574555","type":"journal-article","created":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T19:14:29Z","timestamp":1782846869000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a High\u2010Resolution Melting (HRM)\u2010Based Multiplex Real\u2010Time PCR Assay of PEDV, TGEV, PoRV, PDCoV, PRV, and PRRSV"],"prefix":"10.1155","volume":"2026","author":[{"given":"Fangshan","family":"Wang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Ren","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yawen","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chen","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tairan","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinhai","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2899-8517","authenticated-orcid":false,"given":"Qinye","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"e_1_2_13_1_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2020.198045"},{"key":"e_1_2_13_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2014.08.018"},{"key":"e_1_2_13_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2015.09.021"},{"key":"e_1_2_13_4_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid1801.111259"},{"key":"e_1_2_13_5_2","doi-asserted-by":"publisher","DOI":"10.1002\/prca.201700143"},{"key":"e_1_2_13_6_2","doi-asserted-by":"publisher","DOI":"10.3390\/v9030048"},{"key":"e_1_2_13_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2016.05.028"},{"key":"e_1_2_13_8_2","doi-asserted-by":"publisher","DOI":"10.1111\/tbed.13385"},{"key":"e_1_2_13_9_2","doi-asserted-by":"publisher","DOI":"10.3390\/vetsci9110634"},{"key":"e_1_2_13_10_2","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2024.2332653"},{"key":"e_1_2_13_11_2","doi-asserted-by":"publisher","DOI":"10.1556\/004.2018.030"},{"key":"e_1_2_13_12_2","doi-asserted-by":"publisher","DOI":"10.3390\/v9010019"},{"key":"e_1_2_13_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2019.04.001"},{"key":"e_1_2_13_14_2","doi-asserted-by":"publisher","DOI":"10.1080\/21505594.2020.1771980"},{"key":"e_1_2_13_15_2","doi-asserted-by":"publisher","DOI":"10.7416\/ai.2017.2153"},{"key":"e_1_2_13_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2025.343994"},{"key":"e_1_2_13_17_2","doi-asserted-by":"publisher","DOI":"10.1002\/mbo3.1272"},{"key":"e_1_2_13_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2015.08.012"},{"key":"e_1_2_13_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mcp.2018.03.004"},{"key":"e_1_2_13_20_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12917-015-0364-2"},{"key":"e_1_2_13_21_2","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2023.1158585"},{"key":"e_1_2_13_22_2","doi-asserted-by":"publisher","DOI":"10.3390\/v11020185"},{"key":"e_1_2_13_23_2","doi-asserted-by":"publisher","DOI":"10.3390\/vetsci12090874"},{"key":"e_1_2_13_24_2","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2025.1634353"},{"key":"e_1_2_13_25_2","doi-asserted-by":"publisher","DOI":"10.1080\/01652176.2023.2272188"},{"key":"e_1_2_13_26_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms26178731"},{"key":"e_1_2_13_27_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms13071573"},{"key":"e_1_2_13_28_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.prevetmed.2025.106627"},{"key":"e_1_2_13_29_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00126-18"},{"key":"e_1_2_13_30_2","doi-asserted-by":"crossref","DOI":"10.3389\/fmicb.2023.1092273","article-title":"Establishment and Application of Multiplex Real-Time PCR for Simultaneous Detection of Four Viruses Associated With Porcine reproductive failure","volume":"14","author":"Chen Y.","year":"2023","journal-title":"Frontiers in Microbiology"},{"key":"e_1_2_13_31_2","doi-asserted-by":"crossref","DOI":"10.3389\/fmicb.2022.1049178","article-title":"Multiplex High-Resolution Melting Assay for Simultaneous Detection of Five Key Bacterial Pathogens in Urinary Tract Infections: A Pilot Study","volume":"13","author":"Kafi H.","year":"2022","journal-title":"Frontiers in Microbiology"},{"key":"e_1_2_13_32_2","doi-asserted-by":"crossref","DOI":"10.1186\/s12866-022-02558-2","article-title":"Multiplex Detection of Five Common Respiratory Pathogens From Bronchoalveolar Lavages Using High Resolution Melting Curve Analysis","volume":"22","author":"Ghorbani J.","year":"2022","journal-title":"BMC Microbiology"},{"key":"e_1_2_13_33_2","doi-asserted-by":"crossref","DOI":"10.3390\/v14071355","article-title":"Point-of-Care 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