Establishment of SYBR Green I Real-Time PCR for Detection of Streptococcus agalactiae in Aquaculture Waters

Abstract

Streptococcus agalactiae has a serious negative impact on tilapia aquaculture, and rapid detection of trace S. agalactiae in aquaculture waters and timely and effective sterilization measures could significantly reduce the probability of its outbreak in tilapia farming. Here, we established a fluorescence quantitative detection method of S. agalactiae in tilapia aquaculture waters based on the Streptococcus agalactiae CAMP factor (cfb) (GU217532.1) sequence of its CAMP factor. The results showed that the Ct value and logarithm of plasmid copy number presented a good linear relationship (y = −3.49x + 38.78; R2 = 0.997) in the plasmid concentration range of 1.57 × 102−1.57 × 109 copies/μL, and the lowest concentration for sensitive detection of the established method was 1.57 × 102 copies/μL. The method exhibited high specificity for the detection of S. agalactiae and generated negative results when using the DNA of Aeromonas veronii, Staphylococcus epidermidis, Aeromonas hydrophila, and Edwardsiella tarda as templates for qPCR. Intra- and inter-group repeated experiments produced variation coefficients lower than 2%, indicating high stability and specificity of the method. The method was then used to detect S. agalactiae in two tilapia aquaculture waters. The results showed that the concentration of S. agalactiae was 12 copies/mL and 2 copies/mL in the two water samples, respectively. The method can directly detect trace S. agalactiae in the aquaculture water and facilitate the diagnosis, prevention, and control of S. agalactiae in tilapia aquaculture, which will greatly reduce the probability of large-scale outbreak of S. agalactiae and economic loss.