Development of a real-time PCR assay for detection and quantification ofStreptococcus iniaeusing the lactate permease gene

The aim of this study is the development and evaluation of a rapid and accurate quantitative PCR (qPCR)-based protocol for detection of zoonotic pathogenStreptococcus iniaein bacterial cultures and tissues of diseased fish. For this purpose, the lactate permease-encoding (lldY) gene was selected as a target for the design ofS. iniae-specific primers based on comparative genomic analysis using 45 sequences retrieved from NCBI genome database. Specificity and applicability of these primers were tested using 115 bacterial strains and fish tissues infected withS. iniae. Sensitivity, reproducibility and efficiency of qPCR assay were also determined. The developed qPCR assay showed 100% specificity with pure bacterial cultures or DNA extracted fromS. iniaeor tissues of fish infected with the bacterium. The method has high sensitivity with a detection limit of 1.12 x 10(1)amplicon copies per assay (equivalent to 2 x 10(-9) ng/mu l) using bacterial DNA and of 1.44 x 10(1)gene copies in tissues of fish infected withS. iniae. In conclusion, this qPCR protocol provides an accurate and sensitive alternative for the identification ofS. iniaeand its detection on fish tissues that can be implemented as a routine tool in microbiological laboratories.