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In this study, we developed a real\u2010time PCR assay targeting the cytochrome c oxidase subunit I (<jats:italic>cox1<\/jats:italic>) gene of <jats:italic>M<\/jats:italic>. <jats:italic>avidus<\/jats:italic> to sensitively detect and quantify the parasite in seawater. The assay showed a strong linear correlation between the log copy number of the standard plasmid and Ct values (<jats:italic>R<\/jats:italic><jats:sup>2<\/jats:sup>\u2009=\u20090.9985), achieving an amplification efficiency of 96.26%. The <jats:italic>cox1<\/jats:italic> gene copy number was estimated at 6017\u2009\u00b1\u20092794 copies per cell. The assay specifically detected <jats:italic>M<\/jats:italic>. <jats:italic>avidus<\/jats:italic> (<jats:italic>cox1<\/jats:italic> genotypes 1\u20134) without cross\u2010reacting with <jats:italic>Cryptocaryon irritans<\/jats:italic> or <jats:styled-content style=\"fixed-case\"><jats:italic>Uronema elegans<\/jats:italic><\/jats:styled-content>, identifying as few as two <jats:italic>M<\/jats:italic>. <jats:italic>avidus<\/jats:italic> cells in 1\u2009L of seawater with 100% accuracy (Ct value: 31.95\u2009\u00b1\u20090.78). The noninvasive seawater sampling demonstrates higher sensitivity than invasive tissue sampling. A high density of <jats:italic>M<\/jats:italic>. <jats:italic>avidus<\/jats:italic> (57.81 cells\/10\u2009L) was detected in tanks housing olive flounder with clinical signs of infection, and sporadic detection occurred in both inflow and effluent seawater. The developed real\u2010time PCR assay provides a sensitive, specific and noninvasive method for detecting and quantifying <jats:italic>M<\/jats:italic>. <jats:italic>avidus<\/jats:italic>, enabling improved disease prevention and management in fish farms.<\/jats:p>","DOI":"10.1111\/jfd.14134","type":"journal-article","created":{"date-parts":[[2025,4,23]],"date-time":"2025-04-23T12:58:45Z","timestamp":1745413125000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development of a Real\u2010Time <scp>PCR<\/scp> Assay for Noninvasive Detection of <scp><i>Miamiensis avidus<\/i><\/scp> in Olive Flounder Aquaculture"],"prefix":"10.1111","volume":"48","author":[{"given":"Jin\u2010Young","family":"Kim","sequence":"first","affiliation":[{"name":"Department of Aqualife Medicine Chonnam National University  Yeosu Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6801-8635","authenticated-orcid":false,"given":"Su\u2010Mi","family":"Shin","sequence":"additional","affiliation":[{"name":"Department of Aqualife Medicine Chonnam National University  Yeosu Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Se Ryun","family":"Kwon","sequence":"additional","affiliation":[{"name":"Department of Aquatic Life Medical Sciences Sunmoon\u00a0University  Asan Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9171-6543","authenticated-orcid":false,"given":"Sung\u2010Ju","family":"Jung","sequence":"additional","affiliation":[{"name":"Department of Aqualife Medicine Chonnam National University  Yeosu Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,4,23]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12562\u2010016\u20101016\u20106"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/are.12366"},{"key":"e_1_2_9_4_1","first-page":"105","article-title":"The Effect of Temperature on Scuticociliatida Miamiensis avidus Proliferation, and to Mortality of Infected Olive Flounder Paralichthys olivaceus","volume":"22","author":"Bae M. 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