Abstract:Streptococcus agalactiae has become one of the most important emerging pathogens with strong infectivity and virulence, causing high mortalities and large economic losses in tilapia farming industries in China. There are no obvious symptoms in some tilapia infected by S. agalactiae, so it can be difficult to determine if a fish carried the pathogen. Thus, it was necessary to establish an effective method for detection of S. agalactiae in the breeding process of tilapia. Three pairs of specific primers were designed based on the conserved sequence of hylB, ponA, and cfb genes encoding hyaluronidase, penicillin binding protein, and CAMP factor of S. agalactiae published in GenBank, respectively. After the optimization of the reaction conditions and reaction system of multiplex PCR, the triple PCR method based on the three virulence genes was developed for detection of S. agalactiae isolated from tilapia. Furthermore, the established method was applied to detect tissue samples of tilapia collected from different farming areas in Guangdong Province. The detection result of S. agalactiae only amplified three specific bands, however there were no bands in the host and the other common bacterial pathogen strains in aquaculture, which indicated that the method had good specificity. The dynamic range of template concentration of S. agalactiae was 7.24×10-5~5.65 ng/μl. Sensitivity tests showed that the detection limit of the S. agalactiae in genomic DNA was 1.81×10-3 ng/μl, with a higher sensitivity. The positive rate for detection of 188 tissue samples isolated from tilapia using the triple PCR method was consistent with the positive rate by the conventional bacterial identification method. The triple PCR method was evaluated with the conventional method as the standard; diagnostic sensitivity (Dse) and diagnostic specificity (Dsp) were 100%. In summary, the results showed that the triple PCR method not only improved the accuracy and sensitivity of the detection, but also detected three virulence genes of S. agalactiae in the same reaction system. Therefore, this method provided a rapid, accurate, and efficient detection technology for monitoring S. agalactiae infection in harmless aquatic products and early warning of aquaculture diseases.
