Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate)

ABSTRACT The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1-5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.

[1]  S. L. Le Blancq,et al.  Ribosomal RNA gene organization in Cryptosporidium parvum. , 1997, Molecular and biochemical parasitology.

[2]  David S. Latchman,et al.  Biochemistry (4th edn) , 1995 .

[3]  Molecular approaches to understanding population dynamics of the toxic dinoflagellate Alexandrium fundyense. , 2002, The Biological bulletin.

[4]  A. Dress,et al.  Multiple DNA and protein sequence alignment based on segment-to-segment comparison. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  R. Guillard,et al.  Culture of Phytoplankton for Feeding Marine Invertebrates , 1975 .

[6]  A. Tremblay,et al.  Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene. , 1992, Gene.

[7]  I. Probert,et al.  Morphological details of the life history of Alexandrium minutum (Dinophyceae) , 2002 .

[8]  D. Anderson,et al.  Molecular evolution of the Alexandrium tamarense 'species complex' (Dinophyceae): Dispersal in the North American and West Pacific regions , 1995 .

[9]  E. Delong,et al.  Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays , 2000, Applied and Environmental Microbiology.

[10]  E. Balech,et al.  The genus Alexandrium Halim (Dinoflagellata) , 1995 .

[11]  M. Elbrächter,et al.  Sequence comparisons link toxic European isolates of Alexandrium tamarense from the Orkney Islands to toxic North American stocks , 1998 .

[12]  E. Berdalet,et al.  DETECTION OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM FUNDYENSE (DINOPHYCEAE) WITH OLIGONUCLEOTIDE AND ANTIBODY PROBES: VARIABILITY IN LABELING INTENSITY WITH PHYSIOLOGICAL CONDITION , 1999 .

[13]  I. Olivieri,et al.  Ribosomal External and Internal Transcribed Spacers: Combined Use in the Phylogenetic Analysis of Medicago (Leguminosae) , 1998, Journal of Molecular Evolution.

[14]  C. Tomas,et al.  Identifying marine phytoplankton , 1997 .

[15]  G. Vasta,et al.  Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer , 2002, Applied and Environmental Microbiology.

[16]  M. Sogin,et al.  The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. , 1988, Gene.

[17]  I. H. Öğüş,et al.  NATO ASI Series , 1997 .

[18]  J. Olsen.  NUCLEIC ACIDS IN ALGAL SYSTEMATICS , 1990 .

[19]  Y. Ishida,et al.  ANALYSIS OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING SEQUENCES OF THE 5.8S RIBOSOMAL DNA AND INTERNAL TRANSCRIBED SPACER REGIONS 1 , 1996 .

[20]  T. Smayda,et al.  Harmful algal blooms: Their ecophysiology and general relevance to phytoplankton blooms in the sea , 1997 .

[21]  M. Giacobbe,et al.  THE LIFE HISTORY OF ALEXANDRIUM TAYLORI (DINOPHYCEAE) , 1999 .

[22]  A. Collins,et al.  Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Camp,et al.  Is the distribution of the toxic dinoflagellate Alexandrium catenella expanding along the NW Mediterranean coast , 2001 .

[24]  B. Reguera,et al.  Preparation of DNA suitable for PCR amplification from fresh or fixed single dinoflagellate cells. , 2001, BioTechniques.

[25]  R. Haugland,et al.  Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation. , 1997, Analytical biochemistry.

[26]  Donald M. Anderson,et al.  Physiological ecology of harmful algal blooms , 1998 .

[27]  R. Wachter,et al.  Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity , 2001, Nature.

[28]  T. Lajeunesse.  INVESTIGATING THE BIODIVERSITY, ECOLOGY, AND PHYLOGENY OF ENDOSYMBIOTIC DINOFLAGELLATES IN THE GENUS SYMBIODINIUM USING THE ITS REGION: IN SEARCH OF A “SPECIES” LEVEL MARKER , 2001 .

[29]  D. Hutchins,et al.  Assessing temporal and spatial variability in Pfiesteria piscicida distributions using molecular probing techniques , 2001 .

[30]  L. Connell.  Rapid identification of marine algae (Raphidophyceae) using three-primer PCR amplification of nuclear internal transcribed spacer (ITS) regions from fresh and archived material , 2002 .

[31]  Roman Marin,et al.  DNA PROBES AND A RECEPTOR‐BINDING ASSAY FOR DETECTION OF PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) SPECIES AND DOMOIC ACID ACTIVITY IN CULTURED AND NATURAL SAMPLES , 1999 .

[32]  Hector Macpherson,et al.  Preservation and storage , 1940 .

[33]  D. Anderson,et al.  Paralytic shellfish poisoning toxins in France linked to a human-introduced strain of Alexandrium catenella from the western Pacific: evidence from DNAand toxin analysis , 2002 .

[34]  Gustaaf M. Hallegraeff,et al.  Transport of diatom and dinoflagellate resting spores in ships' ballast water: implications for plankton biogeography and aquaculture , 1992 .

[35]  S. Menden‐Deuer,et al.  Effect of preservation on dinoflagellate and diatom cell volume, and consequences for carbon biomass predictions , 2001 .

[36]  A. Penna,et al.  Toxicity and ribosomal DNA of the dinoflagellate Dinophysis sacculus (Dinophyta) , 2000 .

[37]  B. Dalrymple.  Cloning and characterization of the rRNA genes and flanking regions from Babesia bovis: use of the genes as strain discriminating probes. , 1990, Molecular and biochemical parasitology.

[38]  Molecular typing of the red-tide dinoflagellate Gonyaulax polyedra in phytoplankton suspensions , 1995 .

[39]  Identifying Marine Phytoplankton , 1998 .

[40]  D. Anderson,et al.  Molecular studies of Dinophysis (Dinophyceae) species from Sweden and North America , 2002 .

[41]  Donald M. Anderson,et al.  Toxic Marine Phytoplankton , 1987 .

[42]  J. Camp,et al.  A recurrent and localized dinoflagellate bloom in a Mediterranean beach , 1999 .

[43]  K. Tangen,et al.  Chapter 3 – Dinoflagellates , 1996 .

[44]  W. Smith,et al.  Culture of Marine Invertebrate Animals , 1975, Springer US.

[45]  O. Holm‐Hansen.  Algae: Amounts of DNA and Organic Carbon in Single Cells , 1969, Science.

[46]  P. Walsh,et al.  Simultaneous Amplification and Detection of Specific DNA Sequences , 1992, Bio/Technology.

[47]  V. Nene,et al.  Evidence for two single copy units in Theileria parva ribosomal RNA genes. , 1994, Molecular and biochemical parasitology.

[48]  M. Hershkovitz,et al.  Deep-level diagnostic value of the rDNA-ITS region. , 1996, Molecular biology and evolution.

[49]  Donald M. Anderson,et al.  Turning back the harmful red tide , 1997, Nature.

[50]  I. Karunasagar,et al.  Polymerase Chain Reaction in Detection of Gymnodinium mikimotoi and Alexandrium minutum in Field Samples from Southwest India , 2001, Marine Biotechnology.

[51]  J. Camp,et al.  High Resolution Spatio-temporal Detection of Potentially Harmful Dinoflagellates in Confined Waters of the NW Mediterranean , 2001 .

[52]  P. Gentien,et al.  Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium, Dinophysis, and Karenia) from French coasts. , 2002, Protist.

[53]  J. Burkholder,et al.  Development of Real-Time PCR Assays for Rapid Detection of Pfiesteria piscicida and Related Dinoflagellates , 2000, Applied and Environmental Microbiology.

[54]  R. Zardoya,et al.  MORPHOSPECIES VS. GENOSPECIES IN TOXIC MARINE DINOFLAGELLATES: AN ANALYSIS OF GYMNODZNIUM CATENATUM/GYRODINIUM IMPUDICUM AND ALEXANDRIUM MINUTUM/A. LUSITANICUM USING ANTIBODIES, LECTINS, AND GENE SEQUENCES 1 , 1995 .

[55]  G. Lenaers,et al.  A molecular phylogeny of dinoflagellate protists (Pyrrhophyta) inferred from the sequence of 24S rRNA divergent domains D1 and D8 , 2005, Journal of Molecular Evolution.

[56]  Yasuwo Fukuyo,et al.  Harmful and toxic algal blooms , 1996 .

[57]  L. Fritz,et al.  A RAPID SIMPLE TECHNIQUE UTILIZING CALCOFLUOR WHITE M2R FOR THE VISUALIZATION OF DINOFLAGELLATE THECAL PLATES 1 , 1985 .

[58]  M. Magnani,et al.  IDENTIFICATION OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING PCR AND rDNA‐TARGETED PROBES , 1999 .

[59]  Donald Anderson.  Toxic algal blooms and red tides : a global perspective. , 1989 .