, Volume 695, Issue 1, pp 57–72 | Cite as

The application of oligonucleotide probes and microarrays for the identification of freshwater diatoms

  • Marco Berzano
  • Stefania Marcheggiani
  • Silvia Rombini
  • Roberto Spurio


Diatoms are major contributors to global carbon fixation and constitute a significant portion of biofilms found in lotic ecosystems. Despite their widespread abundance and the fact that extensive studies have been performed on morphological features of frustules, molecular tools for the identification of diatoms are not commonly available. This study focuses on the development of oligonucleotide probes for the detection of diatom species relevant to water quality assessment. The selected panel of diatoms covers all the species found in water of varying quality from the rivers of central-East Apennine (Italy). Small subunit rRNA-targeted probes were applied to a microarray platform as well as to a new technique termed Primer–Probe, with the aim of obtaining a molecular tool suitable for accurate identification of both single and mixed species diatom populations. The Primer–Probe technique together with dot-blot assays proved to be ideal for the preliminary screening of a large set of DNA oligonucleotides designed by ARB software. It was shown that microarrays, as a promising technology for rapid and simultaneous detection of a wide range of species-specific genetic markers, can be adapted to monitor changes within a diatom community. It is suggested that microarrays will provide a molecular basis for microbial identification to support standard microscopy techniques used by ecologists and environmental scientists for monitoring water quality.


Freshwater diatoms Water quality PCR Microarrays Biodiversity 



This study was supported by the EU-project MICROPAD, Grant number QLK3-CT-2002-01939. The authors thank Laura Mancini for her support in the use of ecological indicators and for the identification of freshwater diatoms, Mariacristina Torrisi for her helpful explanations on the use of EPI-D and Mohamed Ali Haider for his contribution to probe design at the early stages of this study. We also acknowledge the late David B. Czarnecki for his helpful advice and thank both Tom Murphy and Cynthia L. Pon for critical reading of the manuscript.

Supplementary material

10750_2012_1112_MOESM1_ESM.doc (87 kb)
Supplementary material 1 (DOC 87 kb)


  1. Amann, R. & W. Ludwig, 2000. Ribosomal RNA-targeted nucleic acid probes for studies in microbial ecology. FEMS Microbiology Review 24: 555–565.CrossRefGoogle Scholar
  2. Amann, R. I., B. J. Binder, R. J. Olson, S. W. Chisholm, R. Devereux & D. A. Stahl, 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Applied and Environmental Microbiology 56: 1919–1925.PubMedGoogle Scholar
  3. Ashelford, K. E., A. J. Weightman & J. C. Fry, 2002. PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Research 30: 3481–3489.PubMedCrossRefGoogle Scholar
  4. Besse-Lototskaya, A. A., P. F. M. Verdonschot, M. Coste & B. Van de Vijver, 2011. Evaluation of European diatom trophic indices. Ecological Indicators 11: 456–467.CrossRefGoogle Scholar
  5. Call, D. R., M. K. Boruckia & F. J. Loge, 2003. Detection of bacterial pathogens in environmental samples using DNA microarrays. Journal of Microbiological Methods 53: 235–243.PubMedCrossRefGoogle Scholar
  6. Castiglioni, B., E. Rizzi, A. Frosini, M. A. Mugnai, S. Ventura, K. Sivonen, P. Rajaniemi, A. Rantala, A. Wilmotte, C. Boutte, C. Consolandi, R. Bordoni, A. Mezzelani, E. Busti, L. Rossi-Bernardi, C. Battaglia & G. de Bellis, 2002. Application of an universal DNA microarray to cyanobacterial diversity assessment. Minerva Biotechnologica 14: 253–257.Google Scholar
  7. Clement, B. G., L. E. Kehl, K. L. DeBord & C. L. Kitts, 1998. Terminal Restriction Fragment Patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. Journal of Microbiological Methods 31: 135–142.CrossRefGoogle Scholar
  8. Dell’Uomo, A., 1996. Assessment of water quality of an Apennine river as a pilot study for diatom-based monitoring of Italian watercourses. In Whitton, B. A. & E. Rott (eds), Proceedings of International Symposium “Use of Algae for Monitoring Rivers II”. Institut für Botanik, Universität Innsbruck, Innsbruck: 65–72.Google Scholar
  9. Dell’Uomo, A., 2004. L’Indice Diatomico di Eutrofizzazione/Polluzione (EPI-D) nel monitoraggio delle acque correnti. Linee Guida. APAT, CTN AIM, Roma, Firenze: 101 pp.Google Scholar
  10. Dell’Uomo, A., A. Pensieri & D. Corradetti, 1999. Diatomées épilithiques du fleuve Esino (Italie centrale) et leur utilisation pour l’évaluation de la qualité biologique de l’eau. Cryptogamie, Algologie 20: 253–269.CrossRefGoogle Scholar
  11. Descy, J. P. & M. Coste, 1991. A test of methods for assessing water quality based on diatoms. Verhandlungen der International Vereinigung für Limnologie 24: 2112–2116.Google Scholar
  12. Elwood, H. J., G. J. Olsen & M. L. Sogin, 1985. The small-subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata. Molecular Biology and Evolution 2: 399–410.PubMedGoogle Scholar
  13. Evans, K. M., A. H. Wortley & D. G. Mann, 2007. An assessment of potential diatom ‘‘Barcode’’ genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta). Protist 158: 349–364.PubMedCrossRefGoogle Scholar
  14. Galluzzi, L., A. Cegna, S. Casabianca, A. Penna, N. Saunders & M. Magnani, 2011. Development of an oligonucleotide microarray for the detection and monitoring of marine dinoflagellates. Journal of Microbiological Methods 84: 234–242.PubMedCrossRefGoogle Scholar
  15. Gescher, C., K. Metfies, S. Frickenhaus, B. Knefelkamp, K. H. Wiltshire & L. K. Medlin, 2008. Feasibility of assessing the community composition of prasinophytes at the Helgoland Roads sampling site with a DNA microarray. Applied and Environmental Microbiology 74: 5305–5316.PubMedCrossRefGoogle Scholar
  16. Guillard, R. R. L., 1975. Culture of phytoplankton for feeding marine invertebrates. In Smith, W. L. & M. H. Chanley (eds), Culture of Marine Invertebrate Animals. Plenum Press, New York: 22–60.Google Scholar
  17. Hamsher, S. E., K. M. Evans, D. G. Mann, A. Poulíčková & G. W. Saunders, 2011. Barcoding diatoms: exploring alternatives to COI-5P. Protist 162: 405–422.PubMedCrossRefGoogle Scholar
  18. Heller, R. A., M. Schena, A. Chai, D. Shalon, T. Bedilion, J. Gilmore, D. E. Woolley & R. W. Davis, 1997. Discovery and analysis of inflammatory disease-related genes using cDNA microarrays. Proceedings of the National Academy of Sciences USA 94: 2150–2155.CrossRefGoogle Scholar
  19. Kelly, J. J., S. Siripong, J. McCormack, L. R. Janus, H. Urakawa, S. El., Fantroussi, P. A. Noble, L. Sappelsa, B. E. Rittmann & D. A. Stahl, 2005. DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples. Water Research 39: 3229–3238.PubMedCrossRefGoogle Scholar
  20. Krammer, K. & H. Lange-Bertalot, 1986–1991. Bacillariophyceae. 1. Teil: Naviculaceae, 876 pp.; 2. Teil: Bacillariaceae, Epithemiaceae, Surirellaceae, 596 pp.; 3. Teil: Centrales, Fragilariaceae, Eunotiaceae, 576 pp.; 4. Teil: Achnanthaceae. Kritische Ergänzungen zu Navicula (Lineolatae) und Gomphonema, 437 pp. In Ettl, H., J. Gerloff, H. Heynig & D. Mollenhauer (eds), Süsswasserflora von Mitteleuropa. Gustav Fischer Verlag, Stuttgart.Google Scholar
  21. Loy, A., R. Arnold, P. Tischler, T. Rattei, M. Wagner & M. Horn, 2008. ProbeCheck—a central resource for evaluating oligonucleotide probe coverage and specificity. Environmental Microbiology 10: 2894–2896.PubMedCrossRefGoogle Scholar
  22. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, I. Yadhukumar, A. Buchner, T. Lai, S. Steppi, G. Jobb, W. Förster, I. Brettske, S. Gerber, A. W. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. König, T. Liss, R. Lüssmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode & K. Schleifer, 2004. ARB: a software environment for sequence data. Nucleic Acids Research 32: 1363–1371.PubMedCrossRefGoogle Scholar
  23. McGrath, K. C., R. Mondav, R. Sintrajaya, B. Slattery, S. Schmidt & P. M. Schenk, 2010. Development of an environmental functional gene microarray for soil microbial communities. Applied and Environmental Microbiology 76: 7161–7170.PubMedCrossRefGoogle Scholar
  24. Medlin, L., H. J. Elwood, S. Stickel & M. L. Sogin, 1988. The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding region. Gene 71: 491–499.PubMedCrossRefGoogle Scholar
  25. Metfies, K. & L. Medlin, 2005. Ribosomal RNA probes and microarrays: their potential use in assessing microbial biodiversity. Methods in Enzymology 395: 258–278.PubMedCrossRefGoogle Scholar
  26. Metfies, K., M. Berzano, C. Mayer, P. Roosken, C. Gualerzi, L. Medlin & G. Muyzer, 2005. An optimized protocol for the identification of diatoms, flagellated algae and pathogenic protozoa with phylochips. Molecular Ecology Notes 7: 925–936.CrossRefGoogle Scholar
  27. Muyzer, G. G., 1999. DGGE/TGGE a method for identifying genes from natural ecosystems. Current Opinion in Microbiology 2: 317–322.PubMedCrossRefGoogle Scholar
  28. Nguyen, T. N. M., M. Berzano, C. O. Gualerzi & R. Spurio, 2011. Development of molecular tools for the detection of freshwater diatoms. Journal of Microbiological Methods 84: 33–40.PubMedCrossRefGoogle Scholar
  29. Patrick, R., 1961. A study of the numbers and kinds of species found in the rivers of Eastern United States. Proceedings of the Academy of Natural Sciences of Philadelphia 113: 215–258.Google Scholar
  30. Peplies, J., C. Lachmund, F. O. Glockner & W. Manz, 2006. A DNA microarray platform based on direct detection of rRNA for characterization of freshwater sediment-related prokaryotic communities. Applied and Environmental Microbiology 72: 4829–4838.PubMedCrossRefGoogle Scholar
  31. Pernthaler, J., F. O. Glöckner, W. Schonhuber & R. Amann, 2001. Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes. In Paul, J. H. (ed.), Methods in Microbiology, Vol. 30., Marine Microbiology Academic Press, San Diego: 207–226.Google Scholar
  32. Porter, J. & R. W. Pickup, 2000. Nucleic acid-based fluorescent probes in microbial ecology: application of flow cytometry. Journal of Microbiological Methods 42: 75–79.PubMedCrossRefGoogle Scholar
  33. Prygiel, J. & M. Coste, 2000. Guide méthodologique pour la mise en oeuvre de l’Indice Biologique Diatomées. NF T 90-354. Agences de l’Eau-Cemagref Bordeaux, Douai: 134 pp.Google Scholar
  34. Rimet, F., H.-M. Cauchie, L. Hoffmann & L. Ector, 2005. Response of diatom indices to simulated water quality improvements in a river. Journal of Applied Phycology 17: 119–128.CrossRefGoogle Scholar
  35. Rott, E., 1991. Methodological aspects and perspectives in the use of periphyton for monitoring and protecting rivers. In Whitton, B. A., E. Rott & G. Friedrich (eds), Use of Algae for Monitoring Rivers. Institut für Botanik, Universität Innsbruck, Innsbruck: 9–16.Google Scholar
  36. Rychlik, W., W. J. Spencer & R. E. Rhoads, 1990. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18: 6409–6412.PubMedCrossRefGoogle Scholar
  37. Sambrook, J. & D. W. Russell, 2001. Molecular Cloning. A Laboratory Manual. CSHL Press, Cold Spring Harbor, NY.Google Scholar
  38. Schena, M., D. Shalon, R. W. Davis & P. O. Brown, 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270: 467–470.PubMedCrossRefGoogle Scholar
  39. Schwieger, F. & C. C. Tebbe, 1998. A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based microbial community analysis. Applied and Environmental Microbiology 64: 4870–4876.PubMedGoogle Scholar
  40. Singh-Gasson, S., R. D. Green, Y. Yue, C. Nelson, F. Blattner, M. R. Sussman & F. Cerrina, 1999. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nature Biotechnology 17: 974–978.PubMedCrossRefGoogle Scholar
  41. Sládeček, V., 1986. Diatoms as indicators of organic pollution. Acta Hydrochimica et Hydrobiologica 14: 555–566.CrossRefGoogle Scholar
  42. Stevenson, R. J. & Y. Pan, 1999. Assessing environmental conditions in rivers and streams with diatoms. In Stoermer, E. F. & J. P. Smol (eds), The Diatoms: Application for the Environmental and Earth Sciences. Cambridge University Press, Cambridge: 11–40.Google Scholar
  43. Tison, J., J.-L. Giraudel & M. Coste, 2008. Evaluating the ecological status of rivers using an index of ecological distance: an application to diatom communities. Ecological Indicators 8: 285–291.CrossRefGoogle Scholar
  44. Werner, D., 1977. The Biology of Diatoms. Blackwell Scientific Publications, Oxford.Google Scholar
  45. Wong, H. C., C. C. Liu, T. M. Pan, T. K. Wang, C. L. Lee & Y. C. Shih, 1999. Molecular typing of Vibrio parahaemolyticus isolates, obtained from patients involved in food poisoning outbreaks in Taiwan by Random Amplified Polymorphic DNA analysis. Journal of Clinical Microbiology 37: 1809–1812.PubMedGoogle Scholar
  46. Zelinka, M. & P. Marvan, 1961. Zur Präzisierung des biologischen Klassifikation der Reinheit fliessender Gewässer. Archiv fur Hydrobiologie 57: 389–407.Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Marco Berzano
    • 1
    • 2
  • Stefania Marcheggiani
    • 2
    • 3
  • Silvia Rombini
    • 2
  • Roberto Spurio
    • 2
  1. 1.Department of Agriculture, Food and the Marine, Central Veterinary Research LaboratoryCelbridgeIreland
  2. 2.Laboratory of Molecular Biology and Microbial Biotechnologies, School of Biosciences and BiotechnologiesUniversity of CamerinoCamerinoItaly
  3. 3.Dipartimenti Ambiente e Prevenzione connessaIstituto Superiore di SanitàRomeItaly

Personalised recommendations