title: Quantitative PCR coupled with melt curve analysis for detection of selected pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean sea.
abstract: The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy.
authors.affiliation: Institut de Recerca i Tecnologia Agroalimentàries, Crta. de Poble Nou, Km 5.5, E-43540 San Carlos de la Rapita (Tarragona), Spain. karl.andree@irta.es
authors.forename: Karl B
authors.initials: KB
authors.lastname: Andree
authors.forename: Margarita
authors.initials: M
authors.lastname: Fernández-Tejedor
authors.forename: Laurence M
authors.initials: LM
authors.lastname: Elandaloussi
authors.forename: Sonia
authors.initials: S
authors.lastname: Quijano-Scheggia
authors.forename: Nagore
authors.initials: N
authors.lastname: Sampedro
authors.forename: Esther
authors.initials: E
authors.lastname: Garcés
authors.forename: Jordi
authors.initials: J
authors.lastname: Camp
authors.forename: Jorge
authors.initials: J
authors.lastname: Diogène
mesh_terms: D016000:Cluster Analysis; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D017377:Diatoms / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D020459:Genes, rRNA; D008522:Mediterranean Sea; D008855:Microscopy, Electron, Scanning; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D012623:Seawater / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004275:DNA, Ribosomal; D021903:DNA, Ribosomal Spacer; D012340:RNA, Ribosomal, 5.8S