Monitoring of Alexandrium species in the Mediterranean Sea using a combined filter system–PCR assay detection method

Abstract

A filter system for collecting phytoplankton cells coupled to a target species-specific polymerase chain reaction (PCR) assay was performed on a spatial and temporal series of net and seawater samples as part of the Mediterranean Sea EU project Strategy (EVK-CT-2001-00046). The application of PCR allowed rapid detection of several harmful dinoflagellate species and genera, including Alexandrium spp. In this study, oligonucleotide primers targeting the 5.8S rDNA and ITS regions of the genus Alexandrium and the species A. minutum were developed. Field samples were concentrated onto filter membranes, total DNA was extracted from mixed phytoplankton populations and PCR assays were carried out with specific primers. Qualitative PCR results were compared with light and epifluorescence microscopic examinations. Results indicated that the molecular assay was able to detect harmful target cells at concentrations undetectable by microscopy. Application of this filter PCR assay to seawater samples revealed it to be a sensitive and rapid procedure for routine monitoring of field samples.