title: Evaluation of taxa-specific real-time PCR, whole-cell FISH and morphotaxonomy analyses for the detection and quantification of the toxic microalgae Alexandrium minutum (Dinophyceae), Global Clade ribotype.
abstract: The dinoflagellate genus Alexandrium contains neurotoxin-producing species that have adversely affected the aquaculture industry in many countries. The morphological similarity between Alexandrium species has led to the development of molecular methods for the discrimination, enumeration and monitoring of toxic and nontoxic species. A quantitative real-time PCR assay (qRT-PCR) targeting the internal transcribed spacer 1-5.8S rRNA gene using hybridization probe technology was developed for the potentially toxic species Alexandrium minutum (Global Clade) (GC). The assay was specific with a detection limit of less than one cell equivalent. The assay was used to detect and quantify A. minutum (GC) in seawater samples collected during summer 2007 in Cork Harbour, Ireland. The results were compared with those obtained using whole-cell FISH (WC-FISH) and morphotaxonomy analyses. Alexandrium minutum did not reach high bloom concentrations over the sampling period (maximum of c. 6 x 10(4) cells L(-1)), and the average concentrations determined using qRT-PCR, WC-FISH and morphotaxonomy did not significantly differ in eight of nine comparisons. Regression curves showed positive relationships between the methods; WC-FISH and qRT-PCR slightly under- and overestimated, respectively, the A. minutum concentrations compared with the morphotaxonomy method. The qRT-PCR assay for A. minutum (GC) offers high-throughput sample analysis and may prove suitable for implementation in microalgae monitoring programmes and assist in population dynamics studies of the species.
authors.affiliation: The Martin Ryan Institute, National University of Ireland, Galway, Ireland. nicolas.touzet@nuigalway.ie
authors.forename: Nicolas
authors.initials: N
authors.lastname: Touzet
authors.forename: Evelyn
authors.initials: E
authors.lastname: Keady
authors.forename: Robin
authors.initials: R
authors.lastname: Raine
authors.forename: Majella
authors.initials: M
authors.lastname: Maher
mesh_terms: D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D004141:Dinoflagellida / Q000145:classification* / Q000166:cytology / Q000235:genetics / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008387:Marine Toxins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D012623:Seawater / Q000382:microbiology*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer; D008387:Marine Toxins; D012340:RNA, Ribosomal, 5.8S