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

OBJECTIVE: 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.