{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T08:07:18Z","timestamp":1788941238331,"version":"build-2803163510"},"reference-count":19,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"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":["Journal of Fish Diseases"],"abstract":"ABSTRACT<\/jats:title>\n \n Piscine lactococcosis is an important bacterial disease of farmed fish. The causative agents,\n \n Lactococcus garvieae<\/jats:italic>\n <\/jats:styled-content>\n ,\n Lactococcus petauri<\/jats:italic>\n and\n Lactococcus formosensis<\/jats:italic>\n , are closely related, which complicates species\u2010level identification. We developed a conventional multiplex PCR assay targeting species\u2010specific genes identified by comparative genomic analysis. Average nucleotide identity reassignment of 441 publicly available genome assemblies identified 111\u2009\n \n L. garvieae<\/jats:italic>\n <\/jats:styled-content>\n , 255\u2009\n L. petauri<\/jats:italic>\n and 75\u2009\n \n L. formosensis<\/jats:italic>\n <\/jats:styled-content>\n genomes. Species\u2010specific primers and a\n tuf<\/jats:italic>\n \u2010based\n Lactococcus<\/jats:italic>\n common control were evaluated using\n in silico<\/jats:italic>\n polymerase chain reaction (PCR) against target genomes and 10,460 off\u2010target assemblies representing 474 taxa in 12 genera. Experimental specificity was assessed using six target strains and 19 non\u2010target fish pathogens. Distinct amplicons of 195, 333 and 500\u2009bp were produced for\n \n L. garvieae<\/jats:italic>\n <\/jats:styled-content>\n ,\n L. petauri<\/jats:italic>\n and\n \n L. formosensis<\/jats:italic>\n <\/jats:styled-content>\n , respectively, together with a 132\u2010bp control amplicon. No cross\u2010amplification was observed. All target species were detected in mixed\u2010DNA samples and spiked kidney and spleen homogenates from two fish species. Analytical detection limits were estimated based on microscopic cell counts of bacterial suspensions before DNA extraction and ranged from 0.956 to 8.55 cell equivalents per reaction. This assay represents a rapid, low\u2010cost method for differentiating lactococcosis\u2010causing\n Lactococcus<\/jats:italic>\n species using standard PCR and agarose gel electrophoresis.\n <\/jats:p>","DOI":"10.1111\/jfd.70288","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T07:50:33Z","timestamp":1788940233000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Multiplex Polymerase Chain Reaction Assay for Differentiating Three\n Lactococcus<\/i>\n Species Associated With Piscine Lactococcosis"],"prefix":"10.1111","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-1532-8943","authenticated-orcid":false,"given":"Tomoaki","family":"Yoshino","sequence":"first","affiliation":[{"name":"Pathology Division, Nansei Field Station Fisheries Technology Institute, Japan Fisheries Research and Education Agency Minami\u2010ise, Watarai Mie Japan"},{"name":"Laboratory of Genome Science Tokyo University of Marine Science and Technology Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tomokazu","family":"Takano","sequence":"additional","affiliation":[{"name":"Pathology Division, Nansei Field Station Fisheries Technology Institute, Japan Fisheries Research and Education Agency Minami\u2010ise, Watarai Mie Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5698-7309","authenticated-orcid":false,"given":"Hiroki","family":"Tanno","sequence":"additional","affiliation":[{"name":"Pathology Division, Nansei Field Station Fisheries Technology Institute, Japan Fisheries Research and Education Agency Minami\u2010ise, Watarai Mie Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukihiro","family":"Nakagawa","sequence":"additional","affiliation":[{"name":"Pathology Division, Tamaki Field Station Fisheries Technology Institute, Japan Fisheries Research and Education Agency Tamaki, Watarai Mie Japan"},{"name":"Laboratory of Fish Pathology Tokyo University of Marine Science and Technology Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Matsuura","sequence":"additional","affiliation":[{"name":"Pathology Division, Nansei Field Station Fisheries Technology Institute, Japan Fisheries Research and Education Agency Minami\u2010ise, Watarai Mie Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shogo","family":"Harakawa","sequence":"additional","affiliation":[{"name":"Ehime Fisheries Research Center Ehime Research Institute of Agriculture, Forestry and Fisheries Uwajima Ehime Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5102-6831","authenticated-orcid":false,"given":"Hidehiro","family":"Kondo","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Science Tokyo University of Marine Science and Technology Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2355-3121","authenticated-orcid":false,"given":"Ikuo","family":"Hirono","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Science Tokyo University of Marine Science and Technology Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8890-8229","authenticated-orcid":false,"given":"Keiichiro","family":"Koiwai","sequence":"additional","affiliation":[{"name":"Laboratory of Genome Science Tokyo University of Marine Science and Technology Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9520-8263","authenticated-orcid":false,"given":"Tomomasa","family":"Matsuyama","sequence":"additional","affiliation":[{"name":"Pathology Division, Nansei Field Station Fisheries Technology Institute, Japan Fisheries Research and Education Agency Minami\u2010ise, Watarai Mie 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