Advertisement

Marine Biotechnology

, Volume 7, Issue 5, pp 506–514 | Cite as

Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay

  • Shoko Hosoi-Tanabe
  • Yoshihiko Sako
Article

Abstract

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenellaspecific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarensespecific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.

Keywords

toxic dinoflagellate Alexandrium paralytic shellfish poisoning (PSP) real-time PCR species-specific identification quantification 

Notes

Acknowledgment

We thank Drs. M. Yamaguchi, T. Uchida (Fisheries Research Agency of Japan), S. Yoshimatsu (Kagawa Fisheries Research Institute), M. Ishida (Aichi Fisheries Research Institute), I. Imai (Kyoto University), M. Yoshida (Nagasaki University), and B. Reguera (Spanish Institute of Oceanography) for providing algal strains. We also thank Mr. Baba (Yamaguchi Prefectural Fisheries Experimental Station), Mr. Tamori (Oita Institute of Marine and Fisheries Science), and Dr. M. Yamaguchi for sampling natural seawater. This work was supported in part by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan, and a grant-in-aid for scientific research (13556033) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Adachi M, Sako Y, Ishida Y (1994) Restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer and 5.8S regions in Japanese Alexandrium species (Dinophyceae) J Phycol 30: 857–863CrossRefGoogle Scholar
  2. Adachi M, Sako Y, Ishida Y (1996) Identification of the toxic dinoflagellates Alexandrium catenella and A. tamarense (Dinophyceae) using DNA probes and whole-cell hybridization J Phycol 32: 1049–1052CrossRefGoogle Scholar
  3. Balech E (1995) The Genus Alexandrium Halim (Dinoflagellate), Cork, Ireland: Sherwin Island PressGoogle Scholar
  4. Bowers HA, Tengs T, Glasgow HB Jr, Burkholder JM, Rublee PA, Oldach DW (2000) Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates Appl Environ Microbiol 66: 4641–4648CrossRefPubMedGoogle Scholar
  5. Chen LCM, Edelstein T, McLachlan J (1969) Bonnemaisonia hamifera Hariot in nature and in culture J Phycol 5: 211–220Google Scholar
  6. Cocolin L, Manzano M, Cantoni C, Comi G (1998) Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food J Appl Microbiol 85: 673–677CrossRefPubMedGoogle Scholar
  7. Galluzzi L, Penna A, Bertozzini E, Vila M, Garces E, Magnani M (2004) Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a Dinoflagellate) Appl Environ Microbiol 70: 1199–1206CrossRefPubMedGoogle Scholar
  8. Guiver M, Levi K, Oppenheim BA (2001) Rapid identification of Candida species by Taq Man PCR J Clin Pathol 54: 362–366CrossRefPubMedGoogle Scholar
  9. Heley ST, Cavender JF, Murray TE (1999). Detection of Alexandrium tamarensis by rapid PCR analysis Biotechques 26: 88–91Google Scholar
  10. Holland PM, Abramson RD, Watson R, Gelfand DH (1991). Detection of specific polymerase chain reaction product by utilizing the 5′-3′ exonuclease activity of Thermus aquaticus DNA polymerase Proc Nacl Acad Sci U S A 88: 7276–7280Google Scholar
  11. Lee LG, Connell CR, Bloch W (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes Nuceleic Acids Res 21: 3761–3766Google Scholar
  12. Lenaers G, Scholin CA, Bhaud Y, Saint-Hilarie D, Herzog M (1991) A molecular phylogeny of dinoflagellate protists (Pyrrophyta) inferred from the sequence of 24S ribosomal DNA divergent domains D1 and D8 J Mol Evol 32: 53–63CrossRefPubMedGoogle Scholar
  13. Lofstrom C, Knutsson R, Axelsson CE, Radstrom P (2004) Rapid and specific detection of Salmonella spp. in animal feed samples by PCR after culture enrichment Appl Environ Microbiol 70: 69–75CrossRefPubMedGoogle Scholar
  14. Mauchline TH, Kerry BR, Hirsch PR (2002) Quantification in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating Appl Environ Microbiol 68: 1846–1853CrossRefPubMedGoogle Scholar
  15. Muller JR, Janz S, Goedert JJ, Potter M, Rabkin CS (1995) Persistence of immunoglobulin heavy chain/c-myc recombination-positive lymphocyte clones in the blood of human immunodeficiency virus-infected homosexual men Proc Natl Acad Sci U S A 92: 6577–6581PubMedGoogle Scholar
  16. Oberst RD, Hays MP, Bohra LK, Phebus RK, Yamahiro CT, Paszko-Kolva C, Flood SJA, Sargeant JM, Gillespie JR (1998) PCR-based DNA amplification and presumptive detection of Escherichia coli O157 : H7 with an internal fluorogenic probe and the 5′ nuclease (Taq Man) assay Appl Environ Microbiol 64: 3389–3396PubMedGoogle Scholar
  17. Penna A, Magnani M (1999) Identification of Alexandrium (Dinophyceae) species using PCR and rDNA-targeted probes J Phycol 35: 615–621CrossRefGoogle Scholar
  18. Penna A, Magnani M (2000). A PCR immunoassay method for the detection of Alexandrium (Dinophyceae) species J Phycol 36: 1183–1186CrossRefGoogle Scholar
  19. Sako Y, Kim C-H, Ninomiya H, Adachi M, Ishida Y (1990) Isozyme and cross analysis of mating population in the Alexandrium catenella / tamarense species complex. In: Graneli E, Sundstrom B, Edler L, Anderson D.M., (eds.). Toxic Marine Phytoplankton, NewYork, N.Y.: Elsevier, 320–323Google Scholar
  20. Sako Y, Hosoi-Tanabe S, Uchida (2004) Fluorescence in situ hybridization using rRNA-targeted probes for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and A. catenella J Phycol 40: 598–605Google Scholar
  21. Schantz EJ, Lynch JM, Vayvanda G, Matsumoto K, Rapoport H (1966) The purification and characterization of the poison produced by Gonyaulax catenella in axenic culture Biochemistry 5: 1191–1195CrossRefPubMedGoogle Scholar
  22. Scholin CA, Herzog M, Sogin M, Anderson DM (1994) Identification of group and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae), II: sequences analysis of a fragment of the LSU rRNA gene J Phycol 30: 999–1011CrossRefGoogle Scholar
  23. Sharma VK, Carlson SA, (2000) Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture Appl Environ Microbiol 66: 5472–5476CrossRefPubMedGoogle Scholar
  24. Shin JH, Nolte FS, Holloway BP, Morrison CJ (1999) Rapid identification of up to three Candida species in a single reaction tube by a 5′ exonuclease assay using fluorescent DNA probes J Clin Microbial 37: 165–170Google Scholar
  25. Shumway SE (1990) A review of the effects of algal blooms on shellfish and aquaculture J World Aquacult Soc 21: 65–104Google Scholar
  26. Zhang H, Lin S (2002) Detection and quantification of Pfiesteria piscicida by using the mitochondrial cytochrome b gene Appl Environ Microbiol 68: 989–994CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  1. 1.Laboratory of Marine Microbiology, Division of Applied Biosciences, Graduate School of AgricultureKyoto UniversityKyotoJapan

Personalised recommendations