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A PCR IMMUNOASSAY METHOD FOR THE DETECTION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES
Author(s) -
Penna Antonella,
Magnani Mauro
Publication year - 2000
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1046/j.1529-8817.2000.99149.x
Subject(s) - biology , digoxigenin , biotinylation , microbiology and biotechnology , dinophyceae , immunoassay , primer (cosmetics) , polymerase chain reaction , chromogenic , chromatography , antibody , biochemistry , gene , chemistry , genetics , in situ hybridization , gene expression , ecology , phytoplankton , organic chemistry , nutrient
PCR primers targeting the internal transcribed spacer (ITS)‐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.