Taengphu S.Dong H.T.Meemetta W.Senapin S.Mahidol University2026-02-062026-02-062026-12-01BMC Research Notes Vol.19 No.1 (2026)https://repository.li.mahidol.ac.th/handle/123456789/114756Objective: To develop, optimize, and validate a novel duplex quantitative PCR (qPCR) assay, termed SaSi qPCR, for the simultaneous detection of Streptococcus agalactiae (Sa) and Streptococcus iniae (Si) in fish samples. Results: The SaSi qPCR assay successfully detected DNA from bacterial cultures and fish specimens, including tilapia (Oreochromis sp.) and Asian sea bass (Lates calcarifer). The method achieved a detection limit of 20 copies per reaction for both targets, with no cross-reactivity observed with other tested fish pathogens. The assay was applied to clinical fish specimens and detected S. agalactiae, S. iniae, and co-infections in fish tissues.Biochemistry, Genetics and Molecular BiologyMedicineArticleSCOPUS10.1186/s13104-025-07577-62-s2.0-1050268932431756050041327378