qPCR assay with a two-step melt curve for the detection of Streptococcus dysgalactiae in fish

Streptococcosis is an economically important bacterial disease of fish, caused by species such as Streptococcus agalactiae, S. iniae, and S. dysgalactiae. Although qPCR assays are available for S. agalactiae and S. iniae, no quantitative assay has been established for S. dysgalactiae in aquaculture. Given the emergence of S. dysgalactiae as a cause of mortality in farmed fish and its zoonotic potential, a sensitive and specific detection method is needed to support aquaculture health management. Two published primer sets, targeting the 16–23S rRNA intergenic spacer (ITS) and the ribonucleoside hydrolase (rihC) gene, were evaluated for their efficiency in detecting S. dysgalactiae. A SYBR Green-based qPCR assay was optimized using the superior primer set, with a two-step melt-curve analysis incorporated to ensure reliable result interpretation, given the characteristics of the ITS sequence. The assay was evaluated for analytical sensitivity, specificity against other fish pathogens, and its applicability to naturally infected tilapia samples. The ITS-targeting primers demonstrated higher amplification efficiency compared to the rihC primers and were selected for assay development. The optimized qPCR assay achieved a limit of detection (LOD) of 81.7 copies, equivalent to 100 fg of bacterial DNA per reaction, and exhibited no cross-reactivity with non-target pathogens. Application of the assay to field samples confirmed the presence of S. dysgalactiae in naturally infected tilapia, demonstrating its diagnostic potential. The developed qPCR assay enables molecular detection of S. dysgalactiae in fish. The incorporation of a two-step melt-curve analysis supports interpretation of the results. This method represents the first qPCR protocol for S. dysgalactiae in aquaculture and offers a valuable tool for disease surveillance, diagnosis, and control in aquaculture. Not applicable.