Researchers in Thailand have developed the first quantitative PCR assay designed specifically to detect Streptococcus dysgalactiae in aquaculture, filling a diagnostic gap for an emerging bacterial pathogen of farmed fish. The SYBR Green-based qPCR assay, described in BMC Methods, achieves a limit of detection of 81.7 copies of bacterial DNA, equivalent to 100 femtograms per reaction, and showed no cross-reactivity with a panel of other fish and shrimp pathogens. When applied to tilapia collected from commercial farms, the assay confirmed the presence of the bacterium in naturally infected fish.
Streptococcosis is an economically important bacterial disease of fish caused by several Streptococcus species, including S. agalactiae, S. iniae, and S. dysgalactiae. Infected fish typically display clinical signs such as exophthalmia, or bulging eyes, and erratic swimming behavior, and the disease can progress either to chronic symptoms or to acute mortality. While infections caused by S. agalactiae and S. iniae are the most commonly reported worldwide, S. dysgalactiae has increasingly been recognized as a cause of mortality in farmed amberjack and yellowtail in Japan, cobia in Taiwan, and tilapia in Brazil and Taiwan. The pathogen also carries zoonotic potential, adding to concerns about its spread in aquaculture systems.
Molecular diagnostics have become standard tools for pathogen detection in fish farming, but the options available for S. dysgalactiae have lagged behind those for its better-studied relatives. Quantitative assays exist for S. agalactiae and S. iniae, yet no qPCR protocol had been established for S. dysgalactiae in aquaculture. Existing PCR and qPCR protocols targeting S. dysgalactiae were developed for the livestock sector, and the only fish-oriented methods reported so far have been endpoint PCR assays, including single assays targeting the 16-23S rRNA intergenic spacer region and the manganese-dependent superoxide dismutase gene, plus a multiplex PCR covering Lactococcus garvieae, S. iniae, and S. dysgalactiae. These non-quantitative approaches require extra post-amplification steps such as gel electrophoresis, which increases the time and labor needed for each test.
To build the new assay, the team, led by Panyaporn Chomsri and Watcharachai Meemetta with colleagues at several Thai institutions, evaluated two previously published primer sets. One set targets the 16-23S rRNA intergenic spacer, or ITS region, a multi-copy genomic region that may offer higher analytical sensitivity. The other targets the ribonucleoside hydrolase gene rihC, a single-copy gene expected to provide greater specificity. Both primer sets were tested using genomic DNA from two S. dysgalactiae isolates obtained from tilapia, with reactions run on a Bio-Rad CFX Connect real-time PCR system using a SYBR Green master mix.
The ITS primers clearly outperformed the rihC primers under the tested conditions. With 200 nanograms of genomic DNA template, the ITS set produced an amplification cycle threshold of roughly 14, compared with roughly 28 for the rihC set, a difference the authors attribute to the higher copy number of the ITS region in the S. dysgalactiae genome. Specificity testing against a panel of non-target bacterial species and virus-infected fish tissues reinforced the choice: the ITS-based assay amplified only S. dysgalactiae DNA, while the rihC-based qPCR produced non-specific amplification across all tested samples. The authors note that the hydrolysis probe included in the original rihC assay was omitted because their system is SYBR Green-based, which may have reduced that primer set’s specificity relative to its original probe-based format.
A distinctive complication arose from the ITS amplicon itself. Melt curve analysis, which is essential in SYBR Green assays for verifying that the amplified product is specific, revealed two distinct melting peaks for the ITS product at ramp rates of 0.5 degrees Celsius, at approximately 76.5 and 84.0 degrees Celsius. Gel electrophoresis confirmed a single sharp band, ruling out non-specific products or primer-dimers. The double-peak pattern reflects uneven GC content along the DNA sequence, since GC-rich regions melt at higher temperatures than AT-rich regions. In silico analyses using uMELT Quartz software showed the same two-peak melt profile and uneven GC distribution for the ITS region, whereas the rihC sequence displayed a single peak and relatively uniform GC content, a pattern previously reported for certain human gene amplicons.
Because such a double peak could easily be misread as non-specific amplification, the researchers tested melt curve protocols at three resolutions: 0.5, 1.0, and 1.3 degrees Celsius ramp rates. At medium resolution the two peaks persisted but were less distinct, while at the lowest resolution a single broader peak appeared at approximately 87 degrees Celsius. Their solution is a two-step melt curve incorporated into the final protocol: first a rapid pass from 65 to 95 degrees Celsius at 1.3 degrees Celsius to capture an overall profile, then a slower pass at 0.5 degrees Celsius to resolve the characteristic double peak. This combination preserves the diagnostic melt signature while reducing the risk of misinterpretation.
The optimized reaction uses an annealing temperature of 65 degrees Celsius and a primer concentration of 200 nanomolar, conditions selected after a gradient test from 55 to 65 degrees Celsius showed that the higher concentration lowered cycle threshold values across the temperature range. In analytical sensitivity testing with ten-fold serial dilutions of bacterial DNA, the assay detected as little as 81.7 copies, or 100 femtograms, of S. dysgalactiae DNA per reaction, corresponding to a cycle threshold of 36.14 plus or minus 0.12, which was set as the cut-off for positive results. The standard curve yielded an amplification efficiency of 72.5 percent with a coefficient of determination of 0.997. The authors attribute the efficiency, which falls below the typical 90 to 110 percent range for qPCR, to the uneven GC distribution of the ITS amplicon, which may affect primer binding or polymerase extension.
To assess real-world applicability, the team applied the assay to samples from Nile tilapia and red hybrid tilapia collected in 2022 from six farms in Thailand. Of 70 fish analyzed, 13 tested positive, or 18.5 percent, with detections on three of the six farms and estimated bacterial loads ranging from 2.23 x 10^4 to 3.77 x 10^6 copies per reaction. Although the proportion of positives was higher among clinically affected fish, 11 of 46 or 23.9 percent, than among apparently healthy fish, 2 of 24 or 8.3 percent, Fisher’s exact test found no statistically significant association between clinical signs and detection of the bacterium. The authors suggest this may reflect subclinical infections in healthy fish and clinical disease caused by other streptococcosis-associated pathogens in qPCR-negative cases.
The authors outline several limitations. The protocol does not include an internal control, so users are advised to run a separate qPCR targeting a validated fish housekeeping gene such as beta-actin or elongation factor 1-alpha to verify sample quality and rule out inhibition. The assay also provides no information on genotypic variation of S. dysgalactiae, and the study did not implement randomized sampling or blinded analysis. Larger sample sizes and comparison with independent reference methods will be needed to formally estimate diagnostic sensitivity and specificity. Even so, the researchers present the assay as the first qPCR protocol for S. dysgalactiae in aquaculture and a practical tool for disease surveillance, diagnosis, and control in farmed fish.
Subject of Research: Development of a SYBR Green qPCR assay with a two-step melt curve for detecting Streptococcus dysgalactiae in farmed fish
Article Title: qPCR assay with a two-step melt curve for the detection of Streptococcus dysgalactiae in fish
Article References: Chomsri, P., Meemetta, W., Taengphu, S., Dong, H. T., Senapin, S., & Panphut, W. (2026). qPCR assay with a two-step melt curve for the detection of Streptococcus dysgalactiae in fish. BMC Methods, 3(1), Article 22. https://doi.org/10.1186/s44330-026-00073-8
Image Credits: AI Generated
DOI: 10.1186/s44330-026-00073-8
Keywords: Streptococcus dysgalactiae, qPCR, aquaculture, fish disease, streptococcosis, tilapia, melt curve analysis, molecular diagnostics, SYBR Green, pathogen detection, assay, two-step
News Source: Kristina Jarvis. (October 29, 2026). New qPCR Test Detects Streptococcus dysgalactiae in Farmed Fish. Scienmag.
