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

PCR primers targeting the internal transcribed spacer (TTS)-5.8S rDNA regio ns specific for the genus Alexandrium were used to develop an ELISA assay m ethod to detect and enumerate this genus in cultured isolates. The solid-ph ase ELISA involves the application of a biotinylated labeled primer to targ et 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 wer e hybridized to an anti-digoxigenin antibody conjugated with alkaline phosp hatase, The presence and number of the Alexandrium cells in the samples res ulted in a proportional appearance of color generated by the phosphatase ac tivity in the presence of a chromogenic substrate and measured in a plate r eader. This PCR and immunoassay solid-phase assay proved to be a useful tec hnique to detect the presence of Alexandrium sp, in cultured isolates and s eawater samples.