Detection and quantification of the toxic marine microalgae Karlodinium veneficum and Karlodinium armiger using recombinase polymerase amplification and enzyme-linked oligonucleotide assay

Abstract

Karlodinium is a dinoflagellate responsible for fish-killing events worldwide. In Alfacs Bay (NW Mediterranean Sea), the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. This work presents a method that combines recombinase polymerase amplification (RPA) with an enzyme-linked oligonucleotide assay (ELONA) to identify, discriminate and quantify these two species. The system was characterised using synthetic DNA and genomic DNA, and the specificity was confirmed by cross-reactivity experiments. Calibration curves were constructed using 10-fold dilutions of cultured cells, attaining a limit of detection of around 50,000 cells/L, far below the Karlodinium spp. alert threshold (200,000 cells/L). Finally, the assay was applied to spiked seawater samples, showing an excellent correlation with the spiking levels and light microscopy counts. This approach is more rapid, specific and user-friendly than traditional microscopy techniques, and shows great promise for the surveillance and management of harmful algal blooms.

Keywords: Enzyme-linked oligonucleotide assay (ELONA); Harmful algal bloom (HAB); Karlodinium armiger; Karlodinium veneficum; Recombinase polymerase amplification (RPA); Seawater.