Monitoring of HAB species in the Mediterranean Sea through molecular methods

A qualitative and semi-quantitative polymerase chain reaction(PCR)-based assay was developed for the detection of severalpotentially Harmful Algal Bloom (HAB) species and genera belongingto Dinophyceae, Bacillariophyceae and Raphydophyceae. Oligonucleotideprimers were designed based on Internal Transcribed Spacer (ITS)–5.8Sribosomal DNA (rDNA) sequences available in public databaseor identified in this study. The specificity and sensitivityof the PCR assay were validated using clonal cultures and thennatural seawater samples, as well as the known copy number ofplasmids containing the target ITS–5.8S rDNA regions.A filter system for collecting mixed phytoplankton cells coupledto a target species-specific PCR assay was performed on spatialand temporal series of net and surface seawater samples duringcoastal water monitoring carried out in several localities ofthe Mediterranean Sea. The application of PCR allowed a rapiddetection of various genera and species-specific potential HABtaxa in all examined natural samples. Qualitative and semi-quantitativePCR results obtained from field samples were compared with microscopiclight microscope (LM)] examinations. The two methods gave comparableresults, and the molecular assay was able to detect HAB targetcells at concentrations not detectable by microscopy or thoseof uncertain identity. The highest values of positive detectionof potential HAB taxon presence obtained by PCR assay comparedwith the microscopic examination ranged from 67 to 8.0%.