SaSi qPCR: a novel duplex qPCR assay for rapid and simultaneous detection of Streptococcus agalactiae and Streptococcus iniae in fish

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. 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.