Rapid detection and Quantification of Fish Killing Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) in Environmental Samples Using Real-time PCR

  • Park, Tae-Gyu (Marine Ecology Research Division, National Fisheries Research and Development Institute) ;
  • Kang, Yang-Soon (Marine Ecology Research Division, National Fisheries Research and Development Institute) ;
  • Seo, Mi-Kyung (Marine Ecology Research Division, National Fisheries Research and Development Institute) ;
  • Kim, Chang-Hoon (Department of Aquaculture, Pukyong National University) ;
  • Park, Young-Tae (Marine Ecology Research Division, National Fisheries Research and Development Institute)
  • Published : 2008.12.31


The mixotrophic dinoflagellate Cochlodinium polykrikoides was reported to be linked to major fish kills in Korea and Japan since the 1990s. Rapid and sensitive detection of microalgae has been problematic because morphological identification of dinoflagellates requires light microscopic and scanning electron microscopic observations that are time consuming and laborious compared to real-time PCR. To address this issue, a real-time PCR probe targeting the ITS2 rRNA gene was used for rapid detection and quantification of C. polykrikoides. PCR inhibitors in water column samples were removed by dilution of template DNA for elimination of false-negative reactions. A strong association between cell quantification using real-time PCR and microscopic counts suggests that the real-time PCR assay is an alternative method for cell estimation of C. polykrikoides in environment samples.


  1. Guillard, R.R.L. and J.H. Ryther. 1962. Studies of marine planktonic diatoms. І. Cyclotella nana Hustedt and Detonula confervacea cleve. Can. J. Microbiol., 8, 229-239
  2. Hallegraeff, G.M. 2003. Harmful algal blooms: a global overview. In G.M. Hallegraeff, Anderson, D.M. and Cembella, A.D. (Eds) Manual on Harmful Marine Microalgae. IOC Manuals and Guides, UNESCO, New York, 25-50
  3. Holland, P.M., R.D. Abramson, R. Watson and D. Gelfand. 1991. Detection of specific polymerase chain reaction products by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA, 88, 7276-7280
  4. Iwataki, M., H. Kawami and K. Matsuoka. 2007. Cochlodinium fulvescens sp. nov. (Gymnodiniales, Dinophyceae), a new chain-forming unarmored dinoflagellate from Asian coasts. Phycol. Res., 55, 231-239
  5. Iwataki, M., H. Kawami, K. Mizushima, C.M. Mikulski, G.J. Doucette, Jr. J.R. Relox, A. Anton, Y. Fukuyo and K. Matsuoka. 2008. Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences. Harmful Algae, 7, 271-277
  6. Lin, S., H. Zhang, Y. Hou, L. Miranda and D. Bhattacharya. 2006. Development of a dinoflagellate-oriented PCR primer set leads to detection of picoplanktonic dino-flagellates from Long Island Sound. Appl. Environ. Microbiol., 72, 5626-5630
  7. NFRDI. 2007. Data on red tides. Retrived from http:// in 2007
  8. Matsuoka, K., M. Iwataki and H. Kawami. 2008. Mor-phology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae). Harmful Algae, 7, 261-270
  9. Mumford, R.A., K. Walsh, I. Barker and N. Boonham. 2000. Detection of potato mop top virus and tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase chain reaction assay. Phytopathology, 90, 448-453
  10. Park, T.G., M.F. de Salas, C.J.S. Bolch and G.M. Hallegraeff. 2007. Development of a real-time PCR probe for quantification of the heterotrophic dino-flagellate Cryptoperidiniopsis brodyi (Dinophyceae) in environmental samples. Appl. Environ. Microbiol., 73, 2552-2560
  11. Park, T.G., G.H. Park, Y.T. Park, Y.S. Kang, H.M. Bae, C.H. Kim, H.J. Jeong and Y. Lee. 2008. Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes. Harmful Algae (In press)
  12. Schena, L. and A. Ippolito. 2003. Rapid and sensitive detection of Rosellinia necatrix in roots and soils by real time Scorpion-PCR. J. Plant Pathol., 85, 15-25
  13. Schűtt, F. 1896. 'Peridiniales' in Engler and Prantl, 'Die natűrlichen Pflanzenfamillien'. Leipzig, Engelmann, I, Abt. B, 43 Figs
  14. Smayda, T.J. 1997. What is a bloom? A commentary. Limnol. Oceanogr., 42, 1132-1136
  15. Taylor, F.J.R. 2004. Illumination or confusion? Dino-flagellate molecular phylogenetic data viewed from a primarily morphological standpoint. Phycol. Res., 52, 308-324

Cited by

  1. Growth Response of the Dinoflagellate Akashiwo sanguinea in Relation to Temperature, Salinity and Irradiance, and its Advantage in Species Succession vol.20, pp.1, 2014,