A PCR method immunoassay for the detection of Alexandrium (Dinophyceae) species

PCR primers targeting the internal transcribed spacer (TTS)-5.8S rDNA regions specific for the genus Alexandrium were used to develop an ELISA assay method to detect and enumerate this genus in cultured isolates. The solid-phase ELISA involves the application of a biotinylated labeled primer to target the specific ITS-5.8S rDNA region; the PCR-amplified products, generated in the presence of digoxigenin-11-deoxiuracil triphosphate nucleotide, are captured on the streptavidin-coated microplate. The captured molecules were hybridized to an anti-digoxigenin antibody conjugated with alkaline phosphatase, The presence and number of the Alexandrium cells in the samples resulted in a proportional appearance of color generated by the phosphatase activity in the presence of a chromogenic substrate and measured in a plate reader. This PCR and immunoassay solid-phase assay proved to be a useful technique to detect the presence of Alexandrium sp, in cultured isolates and seawater samples.