q-PCR-based assay for the toxic dinoflagellate Karenia selliformis monitoring along the Tunisian coasts

Karenia selliformis is a marine dinoflagellate responsible for fish-kill events. Its presence has been reported along the Tunisian coasts (south-eastern Mediterranean Sea) since the 1990s. In the present study, a quantitative-PCR assay, based on the internal transcribed spacer (ITS) molecular marker, was developed to detect and quantify K. selliformis in environmental bivalve mollusk samples and in seawater samples. The assay was optimized, and its specificity was confirmed using cross-reactivity experiments against microalgal species commonly found on the Tunisian coasts and/or closely related to K. selliformis. Calibration curves were performed by tenfold dilutions of plasmid DNA harboring target sequence and genomic DNA, attaining a limit of detection of around 5 copies of target DNA per reaction, far below one K. selliformis cell per reaction. The field application of the developed assay showed a powerful detection capability. Thus, the designed assay could contribute to the deployment of in-field diagnostic tools for K. selliformis blooms monitoring.