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PCR Techniques as Diagnostic Tools for the Identification and Enumeration of Toxic Marine Phytoplankton Species

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Algal Toxins: Nature, Occurrence, Effect and Detection

Abstract

Molecular based techniques applied to the marine environment for the rapid, specific and sensitive detection of target toxic microalgae have been developed and performed in many laboratories worldwide. Further, a need to quantify species specific target HAB (Harmful Algal Bloom) cells in natural assemblages avoiding the time consuming traditional methods of the microscopy is basically required. Here, a brief review of the quailtative and quantitative PCR-based methods applied for the monitoring of HAB species in field samples from the Mediterranean Sea is presented.

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References

  • Adachi, M., Sako, Y., and Ishida, Y., 1996, Analysis of Alexandrium (Dinophyceae) species using sequences of the 5.8S ribosomal DNA and internal transcribed spacer regions. J. Phycol. 32: 424-432.

    Article  CAS  Google Scholar 

  • Anderson, D. M., 1995, Identification of harmful algal species using molecular probes: an emerging perspective, in: Harmful marine algal blooms. Lassus, P., Arzul, G., Erard, E., Gentien, P., Marcaillou, C., eds., Lavoisier Sci. Publ., Paris, pp. 3-13.

    Google Scholar 

  • Avise, J. C., 1998, Conservation genetics in the marine realm. J. Hered. 89: 377-382.

    Article  Google Scholar 

  • Bolch, C. J. S., Blackburn, S. I., Hallegraeff, G. M., and Vaillancourt, R. E., 1999, Genetic variation among strains of the toxic dinoflagellate Gymnodinium catenatum (Dino-phyceae). J. Phycol. 35: 356-367.

    Article  Google Scholar 

  • Bolch, C. J. S., 2001, PCR protocols for genetic identification of dinoflagellates directly from single cyst and plankton cells. Phycologia 40: 162-167.

    Google Scholar 

  • Bowers, H. A., Tengs, T., Glasgow, H. B., Burkhoelder, J. M. j., Rublee, P. A., and Oldach, D. W., 2000, Development of real-time PCR assay for rapid detection of Pfiesteria piscicida and related dinoflagellates. Appl. Environ. Microbiol. 66: 4641-4648.

    Article  PubMed  CAS  Google Scholar 

  • Casper, E. T., Paul, J. H., Smith, M. C., and Gray, M., 2004, Detection and Quantification of the Red Tide Dinoflagellate Karenia brevis by Real-Time Nucleic Acid Sequence-Based Amplification. Appl. Environ. Microbiol. 70: 4727-4732.

    Article  PubMed  CAS  Google Scholar 

  • Connell, L., 2002, Rapid identification of marine algae (Raphidophyceae) using three-primer PCR amplification of nuclear internal transcribed spacer (ITS) regions from fresh and archived material. Phycologia 41: 15-21.

    Google Scholar 

  • Coyne, K. J., Handy, S. M., Demir, E., Whereat, E. B., Hutchins, D. A., Portune, K. J., Doblin, M. A., and Cary, S. C., 2005, Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnol. Oceanogr. Methods 3: 381-391.

    CAS  Google Scholar 

  • Dyhrman, S. T., Erdner, D., La Du, J., Galac, M., and Anderson, D. M., 2006, Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae 5: 242-250.

    Article  CAS  Google Scholar 

  • Edvardsen, B., Shalchian-Tabrizi, K., Jakobsen, K. S., Medlin, L. K., Dahl, E., Brubak, S., and Paasche, E., 2003, Genetic variability and molecular phylogeny of Dinophysis species (Dinophyceae) from Norwegian waters inferred from single cell analysis of rDNA. J. Phycol. 39: 395-408.

    CAS  Google Scholar 

  • Evans, K. M., Bates, S. S., Medlin, L. K., and Hayes, P. K., 2004, Microsatellite marker development and genetic variation in the toxic marine diatom Pseudo-nitzschia multi-series (Bacillariophyceae). J. Phycol. 40: 911-920.

    Article  CAS  Google Scholar 

  • Feral, J. P., 2002, How useful are the genetic markers in attempts to understand and manage marine biodiversity? J. Exp. Mar. Biol. Ecol. 268: 121-145.

    Article  CAS  Google Scholar 

  • Galluzzi, L., Penna, A., Bertozzini, E., Vila, M., Garcés, E., and Magnani, M., 2004, Development of Real-Time PCR assay for rapid detection and quantification of A. minutum species (Dinoflagellate). Appl. Environ. Microbiol. 70: 1199-1206.

    Article  PubMed  CAS  Google Scholar 

  • Galluzzi, L., Penna, A., Bertozzini, E., Vila, M., Garcés, E., Giacobbe, M.G., Prioli, S., and Magnani, M., 2005, Development of a qualitative PCR method for the Alexandrium (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis). Harmful Algae 4: 973-983.

    Article  CAS  Google Scholar 

  • Galluzzi, L., Magnani, M., Saunders, N., Harms, C., and Bruce, I. J., 2007, Current mole-cular techniques for the detection of microbial pathogens. Science Progress 90: 29-50.

    Article  PubMed  CAS  Google Scholar 

  • Galluzzi L., Bertozzini E., Penna A., Perini F., Pigalarga A., Graneli, E., and Magnani, M., Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett. Appl. Microbiol. (in press)

    Google Scholar 

  • Godhe, A., Anderson, D. M., and Rehnstam-Holm, A. S., 2002, PCR amplification of micro-algal DNA for sequencing and species identification: studies on fixatives and algal growth stages. Harmful Algae 1: 375-382.

    Article  CAS  Google Scholar 

  • Gosling, E. M., 1994, Speciation and species concepts in the marine environment, in: Gene-Genetics and evolution of aquatic organisms. A. R. Beaumont, ed., Chapman and Hall, London, pp. 1-15.

    Google Scholar 

  • Granéli, E., and Turner, J. T., 2007, An Introduction to Harmful Algae, in: Ecology of Harmful Algae. E. Granéli, J. T. Turner, eds., Springer, Berlin, pp. 3-7.

    Google Scholar 

  • Guillou, L., Nézan, E., Cueff, V., Erard-Le Denn, E., Cambon-Bonavita, M. A., Gentien, P., and Barbier, G., 2002, Genetic diversity and molecular detection of three toxic dino-flagellate genera (Alexandrium, Dinophysis and Karenia) from French coasts. Protist 153: 223-238.

    Article  PubMed  CAS  Google Scholar 

  • Handy, S. M., Hutchins, D. A., Cary, S. C., and Coyne, K. J., 2006, Simultaneous enume-ration of multiple raphidophyte species by quantitative real-time PCR: capabilities and limitations. Limnol. Oceanogr. Methods 4: 193-204.

    Google Scholar 

  • Hershkovitz, M. A., and Lewis, L. A., 1996, Deep-level diagnostic value of the rDNA-ITS coding region. Mol. Biol. Evol. 13: 1276-1295.

    PubMed  CAS  Google Scholar 

  • Hosoi-Tanabe S., and Sako, Y., 2005, Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by Real-Time PCR assay. Mar. Biotechnol. 7: 506-514.

    Article  PubMed  CAS  Google Scholar 

  • John, U., Fensome, R. A., and Medlin, L. K., 2003, The application of a molecular clock based on molecular sequences and the fossil record to explain biogeographic distribu-tions within the Alexandrium tamarense ‘species complex’ (Dinophyceae). J. Mol. Evol. 20: 1015-1027.

    Article  CAS  Google Scholar 

  • John, U., Groben, R., Beszten, B., and Medlin, L., 2004, Utility of amplified fragment length polynrorphisms (AFLP) to analyse genetic structures within the Alexandrium tamarense species complex. Protist 155: 169-179.

    Article  PubMed  CAS  Google Scholar 

  • John, U., Medlin, L. K., and Groben, R., 2005, Development of specific rRNA probes to distinguish between geographic clades of the Alexandrium tamarense species complex. J. Plank. Res. 27: 199-204

    Article  CAS  Google Scholar 

  • Kamikawa, R., Hosoi-Tanabe, S., Nagai, S., Itakura S., and Sako, Y., 2005, Development of a quantification assay for the cysts of the toxic dinoflagellate Alexandrium tamarense using real-time polymerase chain reaction. Fish. Sci. 71: 987-991.

    Article  CAS  Google Scholar 

  • Kamikawa, R., Asai, J., Miyahara, T., Murata, K., Oyama, K., Yoshimatsu, S., Yoshida T., and Sako, Y., 2006, Application of a Real-time PCR Assay to a Comprehensive Method of Monitoring Harmful Algae. Microb. Environ. 21: 163-173.

    Article  Google Scholar 

  • Lange, M., Chen, Y. Q., and Medlin, L. K., 2002, Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes. Eur. J. Phycol. 37: 77-92.

    Article  Google Scholar 

  • Larsen, A., Castberg, T., Sandaa, R. A., Brussaard, C. P. D., Egge, J. K., Heldal, M., Paulino, A., Thyrhaug, R., van Hannen, E. J., and Bratbak, G., 2001, Population dynamics and diversity of phytoplankton, bacteria and viruses in a seawater enclosure. Mar. Ecol. Progr. Ser. 221: 47-57.

    Article  Google Scholar 

  • Litaker, R. W., and Tester, P. A., 2002, Molecular methods for detecting and characterizing harmful phytoplankton, in: Environmental Microbiology. C. J. Hurst, R. L. Crawford, G. R. Knudsen, M. McInerney, L. D. Stetzenbach, eds., 2nd edition, ASM Press, New York, USA, pp. 342-353.

    Google Scholar 

  • Lundholm, N. M. Ø., Kotaki, Y., Hoef-Emden, K., Scholin, C., and Miller, P., 2006, Inter-and intraspecific variation of the Pseudo-nitzschia delicatissima complex (Bacillario-phyceae) illustrated by rRNA probes, morphological data and phylogenetic analyses. J. Phycol. 42: 464-481.

    Article  CAS  Google Scholar 

  • Medlin, L. K., Metfies, K., Mehl, H., Wiltshire, K., and Valentin, K., 2006, Picoeukaryotic plankton diversity at the Helgoland time series site as assessed by three molecular methods. Microb. Ecol. 52: 53-71.

    Article  PubMed  CAS  Google Scholar 

  • Metfies, K., and Medlin, L. K., 2004, DNA microchips for phytoplankton: the fluorescent wave of the future. Nova Hedwigia 79: 321-327.

    Article  Google Scholar 

  • Metfies, K., Huljic, S., Lange, M., and Medlin, L. K., 2005, Electrochemical detection of the toxic dinoflagellate Alexandrium ostenfeldii with a DNA-biosensor. Biosens. Bioelec-tron. 20: 1349-1357.

    Article  CAS  Google Scholar 

  • Miller, P. E., and Scholin, C. A., 2000, On detection of Pseudo-nitzschia (Bacillariophyceae) species using whole cell hybridization: sample fixation and stability. J. Phycol. 36:238-250.

    Article  Google Scholar 

  • Monis, P. T., Giglio, S., Keegan, A. R., and Andrew Thompson, R. C., 2005, Emerging technologies for the detection and genetic characterization of protozoan parasites. Trends Parasitol. 21: 340-346.

    Article  PubMed  CAS  Google Scholar 

  • Not, F., Latasa, M., Marie, D., Cariou, T., Vaulot, D., and Simon, N., 2004, A single species, Micromonas pusilla (Prasinophyceae), dominates the eukaryotic picoplankton in the Western English Channel. Appl. Environ. Microb. 70: 4064-4072.

    Article  CAS  Google Scholar 

  • Orsini, L., Sarno, D., Procaccini, G., Poletti, R., Dahlmann, J., and Montresor, M., 2002, Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: morpho-logy, toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species. Eur. J. Phycol. 37: 247-257.

    Article  Google Scholar 

  • Orsini, L., Procaccini, G., Sarno, D., and Montresor, M., 2004, Multiple rDNA ITS-types within the diatom Pseudo-nitzschia delicatissima (Bacillariophyceae) and their relative abundances across a spring bloom in the Gulf of Naples. Mar. Ecol. Progr. Ser. 271: 87-98.

    Article  CAS  Google Scholar 

  • Palumbi, S. R., 1992, Marine speciation on a small placet. Tree 7: 114-118.

    Google Scholar 

  • Palumbi, S. R., 1994, Genetic divergence, reproductive isolation, and marine speciation. Ann. Rev. Ecol. Syst. 25: 547-572.

    Article  Google Scholar 

  • Park T. G., de Salas, M. F., Bolch, C. J., and Hallegraeff, G. M., 2007, Development of a real-time PCR probe for quantification of the heterotrophic dinoflagellate Crypto-peridiniopsis brodyi (Dinophyceae) in environmental samples. Appl. Environ. Microbiol. 73: 2552-2560.

    Article  PubMed  CAS  Google Scholar 

  • Parsons, M. L., Scholin, C. A., Miller, P. E., Doucette, G. J., Powell, C.L., Fryxell, G.A., Dortch, Q., and Soniat, T.M., 1999, Pseudo-nitzschia species (Bacillariophyceae) in Louisiana coastal waters: molecular probe field trials, genetic variability and domoic acid analyses. J. Phycol. 35: 1368-1378.

    Article  Google Scholar 

  • Penna, A., and Magnani, M., 2000, A PCR immunoassay method for the detection of Alexandrium (Dinophyceae) species. J. Phycol. 36: 1183-1186.

    Article  CAS  Google Scholar 

  • Penna, A., Garcés, E., Vila, M., Giacobbe, M. G., Fraga, S., Luglié, A., Bravo, I., Bertozzini, E., and Vernesi, C., 2005a, Alexandrium catenella (Dinophyceae), a toxic ribotype expanding in the NW Mediterranean Sea. Mar. Biol. 148: 13-23.

    Article  Google Scholar 

  • Penna, A., Vila, M., Fraga, S., Giacobbe, M. G., Andreoni, F., Riobó, P., and Vernesi, C., 2005b, Characterization of Ostreopsis and Coolia (Dinophyceae) isolates in the Western Mediterranean Sea based on morphology, toxicity and internal transcribed spacer 5.8S rDNA sequences. J. Phycol. 41: 212-225.

    Article  CAS  Google Scholar 

  • Penna, A., Bertozzini, E., Batocchi, C., Giacobbe, M. G., Galluzzi, L., Garcés, E., Vila, M., Lugliè, A., and Magnani, M., 2007, Monitoring of HAB species in the Mediterranean Sea through molecular techniques. J. Plank. Res. 29: 19-38.

    Article  CAS  Google Scholar 

  • Popels, L. C., Cary, S. C., Hutchins, D. A., Forbes, R., Pustizzi, F., Gobler, C. J., and Coyne, K. J., 2003, The use of quantitative polymerase chain reaction for the detection and enumeration of the harmful alga Aureococcus anophagefferens in environmental samples along the United States East Coast. Limnol. Oceanogr. Methods 1: 92-102.

    Google Scholar 

  • Raoult, D., Fournier, P. E., and Drancourt, M., 2004, What does the future hold for clinical microbiology? Nat. Rev. Microbiol. 2: 151-159.

    Article  PubMed  CAS  Google Scholar 

  • Round Table Detection and Monitoring of HABs, by Penna, A., Silke, J., and Diogene, J., XI International Conference Harmful Algal Blooms, 14-19 November 2004, Cape Town.

    Google Scholar 

  • Saito, K., Drgon, T., Robledo, J. A. F., Krupatkina, D. N., and Vasta, G. R., 2002, Charac-terization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR based detection assays targeted to the nontranscribed spacer. Appl. Environ. Microb. 68: 5394-5407.

    Article  CAS  Google Scholar 

  • Sako, Y., Hosoi-Tanabe, S., and Uchida, A., 2004, Fluorescence in situ hybridization using rRNA-targeted probes for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. J. Phycol. 40: 598-605.

    Article  CAS  Google Scholar 

  • Scholin, C. A., Herzog, M., Sogin, M., and Anderson, D. M., 1994, Identification of group-and strain-specific genetic markers from globally distributed Alexandrium (Dino-phyceae). II. Sequence analysis of fragments of the LSU rRNA gene. J. Phycol. 30: 999-1011.

    Article  CAS  Google Scholar 

  • Scholin, C. A., Buck, K. R., Britschgi, T., Cangelosi, G., and Chavez, F. P., 1996, Identi-fication of Pseudo-nitzschia australis (Bacillariophyceae) using rRNA-targeted probes in whole cells and sandwich hybridization formats. Phycologia 35: 190-197.

    Article  Google Scholar 

  • Scholin, C. A., Marin, R., Doucette, G. J., Powell, C. L., Haydock, P., Howard, J., and Ray, J., 1999, DNA probes and a receptor-binding assay for detection of Pseudo-nitzschia (Bacillariophyceae) species and domoic acid activity in cultured and natural samples. J. Phycol. 35: 1356-1367.

    Article  CAS  Google Scholar 

  • Simon, N., Campbell, L., Ornolfsdottir, E., Groben, R., Guillou, L., Lange, M., and Medlin, L. K., 2000, Oligonucleotide probes for the identification of three algal groups by dot blot and fluorescent whole-cell hybridisation. J. Euk. Microbiol. 47: 76-84.

    Article  PubMed  CAS  Google Scholar 

  • Tyrrell, J. V., Bergquist, P. R., Bergquist, P. L., and Scholin, C. A., 2001, Detection and enu-meration of Heterosigma akashiwo and Fibrocapsa japonica (Raphydophyceae) using rRNA-targeted oligonucleotide probes. Phycologia 40: 457-467.

    Article  Google Scholar 

  • Van Hannen, E. J., van Agterveld, M. P., Gons, H. J., and Laanbroek, H. J., 1998, Revealing genetic diversity of eukaryotic microorganisms in aquatic environments by denaturing gradient gel electrophoresis. J. Phycol. 34: 206-213.

    Article  CAS  Google Scholar 

  • Vila, M., Giacobbe, M. G., Masó, M., Gangemi, E., Penna, A., Sampedro, N., Azzaro, F., Camp, J., and Galluzzi, L., 2005, A comparative study on recurrent blooms of Alexan-drium minutum in two Mediterranean coastal areas. Harmful Algae 4: 673-695.

    Article  Google Scholar 

  • Zhang, H., and Lin, S., 2005, Development of a cob-18S rRNA gene real-time PCR assay for quantifying Pfiesteria shumwayae in the natural environment. Appl. Environ. Microbiol. 71: 7053-7063.

    Article  PubMed  CAS  Google Scholar 

Readings on PCR Techniques

  • Altshuler, M., 2006, PCR troubleshooting: the essential guide. Caister Academic Press, Norfolk, U.K.

    Google Scholar 

  • Dieffenbach, C. W., and Dveksler, G. S., 2006, PCR Primer: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, New Jork, USA.

    Google Scholar 

  • Edwards, K., Logan, J., and Saunders, N., 2004, Real-Time PCR An essential guide. Horizon Bioscience, Norfolk, U.K.

    Google Scholar 

  • McPherson, M. J., and Møller, S. G., 2006, PCR, 2nd ed., Taylor and Francis, Cornwall, UK.

    Google Scholar 

  • Mullis, K. B., 1990, The unusual origins of the polymerase chain reaction. Sci. Am. 262: 56-65.

    Article  PubMed  CAS  Google Scholar 

  • Tevfik Dorak, M., 2006, Real Time PCR, Taylor and Francis, Cornwall, U.K.

    Google Scholar 

  • White, T. J., 1996, The future of PCR technology: diversification of technologies and applications. Trends Biotechnol. 14: 478-483.

    Article  PubMed  CAS  Google Scholar 

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Penna, A., Galluzzi, L. (2008). PCR Techniques as Diagnostic Tools for the Identification and Enumeration of Toxic Marine Phytoplankton Species. In: Evangelista, V., Barsanti, L., Frassanito, A.M., Passarelli, V., Gualtieri, P. (eds) Algal Toxins: Nature, Occurrence, Effect and Detection. NATO Science for Peace and Security Series A: Chemistry and Biology. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-8480-5_12

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