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Early detection of potentially virulent isolates in fish and environmental samples is essential for effective disease prevention and management, particularly under field conditions where the pathogen may be present at low abundance. In this study, we developed and evaluated the analytical and diagnostic performance of a two\u2010step detection protocol that combines a short alkaline peptone water (APW\u20101) enrichment step with a duplex PCR assay targeting the species\u2010specific\n                    <jats:italic>toxR<\/jats:italic>\n                    gene and the plasmid\u2010associated\n                    <jats:italic>fpcrp<\/jats:italic>\n                    gene, a putative marker linked to increased virulence in some fish\u2010pathogenic vibrios. This marker was detected in a diverse set of publicly available genomes from virulent\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>V.\u2009harveyi<\/jats:italic>\n                    <\/jats:styled-content>\n                    isolates as well as in virulent isolates used in this study. This integrated approach enables reliable detection of\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>V.\u2009harveyi<\/jats:italic>\n                    <\/jats:styled-content>\n                    in complex biological and environmental matrices while simultaneously providing virulence\u2010associated screening. The duplex PCR showed high analytical specificity for\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>V.\u2009harveyi<\/jats:italic>\n                    <\/jats:styled-content>\n                    based on\n                    <jats:italic>toxR<\/jats:italic>\n                    amplification when tested against a panel of closely related\n                    <jats:italic>Vibrio<\/jats:italic>\n                    species and allowed the identification of\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>V.\u2009harveyi<\/jats:italic>\n                    <\/jats:styled-content>\n                    isolates carrying the putative virulence\u2010associated marker. Without enrichment, the limit of detection was approximately 10\n                    <jats:sup>5<\/jats:sup>\n                    \u2009CFU\/g or CFU\/mL in fish tissues and water samples, whereas incorporation of an 8\u2009h APW\u20101 enrichment step improved the analytical sensitivity to around 10\n                    <jats:sup>2<\/jats:sup>\n                    \u2009CFU\/g or CFU\/mL. The diagnostic performance of the complete two\u2010step protocol was evaluated using experimentally spiked samples and field samples from Mediterranean aquaculture settings, including fish tissues and aquatic environments. Overall, this enrichment\u2010duplex PCR assay provides a rapid, sensitive and practical tool for the early detection and risk\u2010oriented monitoring of potentially virulent\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>V.\u2009harveyi<\/jats:italic>\n                    <\/jats:styled-content>\n                    isolates in aquaculture environments, supporting surveillance programmes and control strategies.\n                  <\/jats:p>","DOI":"10.1111\/jfd.70224","type":"journal-article","created":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T10:55:41Z","timestamp":1782298541000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Enrichment\u2010Based Duplex\n                    <scp>PCR<\/scp>\n                    for Early Detection of Potentially Virulent\n                    <scp>\n                      <i>Vibrio harveyi<\/i>\n                    <\/scp>\n                    in Aquaculture"],"prefix":"10.1111","volume":"49","author":[{"given":"Javier","family":"Barriga\u2010Cuartero","sequence":"first","affiliation":[{"name":"University Institute for Biotechnology and Biomedicine Research (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carla","family":"Hern\u00e1ndez\u2010Cabanyero","sequence":"additional","affiliation":[{"name":"University Institute for Biotechnology and Biomedicine Research (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pablo","family":"Ibanyez\u2010Paya","sequence":"additional","affiliation":[{"name":"University Institute for Biotechnology and Biomedicine Research (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmen","family":"Amaro","sequence":"additional","affiliation":[{"name":"University Institute for Biotechnology and Biomedicine Research (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Belen","family":"Fouz","sequence":"additional","affiliation":[{"name":"University Institute for Biotechnology and Biomedicine Research (BIOTECMED) Universitat de Val\u00e8ncia  Burjassot Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,6,24]]},"reference":[{"key":"e_1_2_12_2_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1472\u2010765X.2006.01986.x"},{"key":"e_1_2_12_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0022\u20102836(05)80360\u20102"},{"key":"e_1_2_12_4_1","doi-asserted-by":"publisher","DOI":"10.1079\/9781789243277.0182"},{"key":"e_1_2_12_5_1","doi-asserted-by":"publisher","DOI":"10.1177\/1176934320903288"},{"key":"e_1_2_12_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2009.03.015"},{"key":"e_1_2_12_7_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-319-32674-0"},{"key":"e_1_2_12_8_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1472\u2010765X.2006.01989.x"},{"key":"e_1_2_12_9_1","unstructured":"Barriga\u2010Cuartero J. 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