High-throughput identification and quantification of Vagococcus salmoninarum by SYBR Green I-based real-time PCR combined with melting curve analysis

Abstract

This work describes a primer pair and a high-throughput SYBR Green I-based real-time PCR protocol combined with melting curve analysis for identification and quantification of Vagococcus salmoninarum in bacterial cultures and infected fish tissues. The 16S rRNA gene was selected for the design of the primer pair (SalF and SalR). The sensitivity and specificity of this primer pair were compared with other previously designed for conventional PCR. Although both primer pairs showed 100% specificity using pure bacterial cultures or DNA extracted from bacteria or fish tissues, the primer pairs designed in this study showed the highest sensitivity with a detection limit of 0.034 × 10⁰ amplicon copies per assay (equivalent to 2 × 10^-11 ng/µl, C_q value of 30.49 ± 1.71). The developed qPCR protocol allowed the detection of V. salmoninarum in non-lethal and lethal fish samples with detection levels of 0.17 × 10⁰ gene copies in tissues artificially infected and 0.02 × 10⁰ in tissues of fish experimentally infected with V. salmoninarum. The high sensitivity of the developed method suggests that it could be considered as a useful tool for diagnosis of vagococcosis and the detection of V. salmoninarum in asymptomatic or carrier fish.

Keywords: Vagococcus salmoninarum; diagnosis; melting curve; rainbow trout; real-time polymerase chain reaction.