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To address this challenge, a novel triplex TaqMan quantitative PCR (qPCR) assay was developed for the simultaneous detection of three high\u2010risk agents: Infectious Myonecrosis Virus (IMNV), Decapod Iridescent Virus 1 (DIV1), and Covert Mortality Nodavirus (CMNV). This study not only establishes a robust multiplex platform but also systematically optimises its performance, achieving superior analytical sensitivity and reproducibility compared to existing single\u2010target or duplex methods. The assay demonstrated a limit of detection as low as 10\n                    <jats:sup>1<\/jats:sup>\n                    copies\/\u03bcL for all targets, with excellent inter\u2010 and intra\u2010assay coefficients of variation (CVs) consistently below 3%. Notably, the method effectively overcomes the technical hurdles associated with detecting both RNA (IMNV, CMNV) and DNA (DIV1) viruses within a single reaction, eliminating cross\u2010reactivity and ensuring high specificity. Validated against clinical samples, the established protocol exhibits a concordance rate exceeding 95% with current standard methods, while significantly streamlining the diagnostic workflow. This advancement provides a powerful tool for routine biosecurity monitoring, seed stock certification, and the comprehensive management of emerging shrimp diseases.\n                  <\/jats:p>","DOI":"10.1111\/jfd.70229","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T12:10:56Z","timestamp":1784031056000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Establishment and Application of a Triple TaqMan qPCR Detection Method for IMNV, DIV1, and CMNV"],"prefix":"10.1111","volume":"49","author":[{"given":"Yuzhi","family":"Wang","sequence":"first","affiliation":[{"name":"Laboratory of Pathology and Immunology of Aquatic Animals, KLMME Ocean University of China  Qingdao China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chuang","family":"Cui","sequence":"additional","affiliation":[{"name":"Laboratory of Pathology and Immunology of Aquatic Animals, KLMME Ocean 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