Journal of Applied Phycology

, Volume 30, Issue 4, pp 2435–2445 | Cite as

Dual quantitative PCR assay for identification and enumeration of Karlodinium veneficum and Karlodinium armiger combined with a simple and rapid DNA extraction method

  • Anna Toldrà
  • Karl B. Andree
  • Margarita Fernández-Tejedor
  • Jorge Diogène
  • Mònica Campàs


Karlodinium is a dinoflagellate genus responsible for massive fish mortality events worldwide. It is commonly found in Alfacs Bay (NW Mediterranean Sea), where the presence of two Karlodinium species (K. veneficum and K. armiger) with different toxicities has been reported. Microscopy analysis is not able to differentiate between these two species. Therefore, new and rapid methods that accurately and specifically detect and differentiate these two species are crucial to facilitate routine monitoring, to provide early warnings and to study population dynamics. In this work, a quantitative real-time PCR (qPCR) method to detect and enumerate K. veneficum and K. armiger is presented. The ITS1 region of the ribosomal DNA was used to design species-specific primers. The specificity of the primers together with the melting curve profile provided a reliable qualitative identification and discrimination between the two Karlodinium species. Additionally, a simple and rapid DNA extraction method was used. Standard curves were constructed from 10-fold dilutions of cultured microalgae cells. Finally, the applicability of the assay was tested with field samples collected from Alfacs Bay. Results showed a significant correlation between qPCR determinations and light microscopy counts (y = 2.838 x + 564; R2 = 0.936). Overall, the qPCR method developed herein is specific, rapid, accurate, and promising for the detection of these two Karlodinium species in environmental samples.


Karlodinium veneficum Karlodinium armiger Dinoflagellate Quantitative PCR ITS rDNA DNA extraction 



The authors would like to thank Núria Orra and Rosa Fibla for the initial development of the qPCR methodology as well as María Rey for field sample counting by optical microscopy and her help on culturing of microalgae, José Luis Costa for collecting Karlodinium samples at Alfacs Bay, and Josep Fumadó for kindly helping to test the environmental samples by qPCR.

Funding information

This study received financial support from the Ministerio de Economía, Industria y Competitividad through the SEASENSING (BIO2014-56024-C2-2-R) and PURGA DE MAR (IPT-2011-1707-310000) projects. This study also received support from CERCA Programme/Generalitat de Catalunya. Anna Toldrà received her PhD grant (2015PMF-PIPF-67) from IRTA-Universitat Rovira i Virgili-Banco Santander.

Supplementary material

10811_2018_1446_MOESM1_ESM.docx (14 kb)
ESM 1 (DOCX 14 kb)
10811_2018_1446_MOESM2_ESM.docx (40 kb)
ESM 2 (DOCX 40 kb)
10811_2018_1446_MOESM3_ESM.docx (14 kb)
ESM 3 (DOCX 14 kb)


  1. AlgaeBase; searched on 26 February 2018
  2. Andree KB, Fernandez-Tejedor M, Elandaloussi LM, Quijano-Scheggia S, Sampedro N, Garces E, Camp J, Diogene J (2011) Quantitative PCR coupled with melt curve analysis for detection of selected Pseudo-nitzschia spp. (Bacillariophyceae) from the northwestern Mediterranean Sea. Appl Environ Microbiol 77:1651–1659CrossRefPubMedGoogle Scholar
  3. Battocchi C, Totti C, Vila M, Maso M, Capellacci S, Accoroni S, Rene A, Scardi M, Penna A (2010) Monitoring toxic microalgae Ostreopsis (dinoflagellate) species in coastal waters of the Mediterranean Sea using molecular PCR-based assay combined with light microscopy. Mar Poll Bull 60:1074–1084CrossRefGoogle Scholar
  4. Berge T, Poulsen LK, Moldrup M, Daugbjerg N, Hansen PJ (2012) Marine microalgae attack and feed on metazoans. ISME J 6:1926–1936CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bergholtz T, Daugbjerg N, Moestrup O, Fernandez-Tejedor M (2006) On the identity of Karlodinium veneficum and description of Karlodinium armiger sp nov (Dinophyceae), based on light and electron microscopy, nuclear-encoded LSU rDNA, and pigment composition. J Phycol 42:170–193CrossRefGoogle Scholar
  6. Casabianca S, Perini F, Casabianca A, Battocchi C, Giussani V, Chiantore M, Penna A (2014) Monitoring toxic Ostreopsis cf. ovata in recreational waters using a qPCR based assay. Mar Poll Bull 88:102–109CrossRefGoogle Scholar
  7. Daugbjerg N, Hansen G, Larsen J, Moestrup O (2000) Phylogeny of some of the major genera of dinoflagellates based on ultrastructure and partial LSU rDNA sequence data, including the erection of three new genera of unarmoured dinoflagellates. Phycologia 39:302–317Google Scholar
  8. Eckford-Soper LK, Daugbjerg N (2015a) Development of a multiplex real-time qPCR assay for simultaneous enumeration of up to four marine toxic bloom-forming microalgal species. Harmful Algae 48:37–43CrossRefPubMedGoogle Scholar
  9. Eckford-Soper LK, Daugbjerg N (2015b) Examination of six commonly used laboratory fixatives in HAB monitoring programs for their use in quantitative PCR based on Taqman probe technology. Harmful Algae 42:52–59CrossRefGoogle Scholar
  10. Eckford-Soper LK, Daugbjerg N (2016) A quantitative real-time PCR assay for identification and enumeration of the occasionally co-occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures. J Phycol 52:174–183CrossRefPubMedGoogle Scholar
  11. Erdner DL, Percy L, Keafer B, Lewis J, Anderson DM (2010) A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments. Deep-Sea Res II 57:279–287Google Scholar
  12. Fawley MW, Fawley KP (2004) A simple and rapid technique for the isolation of DNA from microalgae. J Phycol 40:223–225Google Scholar
  13. Fernandez-Tejedor M, Soubrier-Pedreno MA, Furones MD (2004) Acute LD50 of a Gyrodinium corsicum natural population for Sparus aurata and Dicentrarchus labrax. Harmful Algae 3:1–9CrossRefGoogle Scholar
  14. Galluzzi L, Bertozzini E, Penna A, Perini F, Garces E, Magnani M (2010) Analysis of rRNA gene content in the Mediterranean dinoflagellate Alexandrium catenella and Alexandrium taylori: implications for the quantitative real-time PCR-based monitoring methods. J Appl Phycol 22:1–9CrossRefGoogle Scholar
  15. Galluzzi L, Bertozzini E, Penna A, Perini F, Pigalarga A, Graneli E, Magnani M (2008) Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Lett Appl Microbiol 46:261–266CrossRefPubMedGoogle Scholar
  16. 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 70:1199–1206CrossRefPubMedPubMedCentralGoogle Scholar
  17. Garces E, Delgado M, Maso M, Camp J (1999) In situ growth rate and distribution of the ichthyotoxic dinoflagellate Gyrodinium corsicum Paulmier in an estuarine embayment (Alfacs Bay, NW Mediterranean Sea). J Plankton Res 21:1977–1991CrossRefGoogle Scholar
  18. Garces E, Fernandez M, Penna A, Van Lenning K, Gutierrez A, Camp J, Zapata M (2006) Characterization of NW Mediterranean Karlodinium spp. (Dinophyceae) strains using morphological, molecular, chemical, and physiological methodologies. J Phycol 42:1096–1112CrossRefGoogle Scholar
  19. Godhe A, Asplund ME, Harnstrom K, Saravanan V, Tyagi A, Karunasagar I (2008) Quantification of diatom and dinoflagellate biomasses in coastal marine seawater samples by real-time PCR. Appl Environ Microbiol 74:7174–7182CrossRefPubMedPubMedCentralGoogle Scholar
  20. Greco M, Saez CA, Bitonti MB (2014) A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga. PLoS One 9(7):e96470CrossRefPubMedPubMedCentralGoogle Scholar
  21. Guillard RRL (1973) Division rates. In: Stein J (ed) Culture methods and growth measurements. Cambridge University Press, Cambridge, p 289–320Google Scholar
  22. Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can J Microbiol 8:229–239Google Scholar
  23. Guillard RRL, Hargraves PE (1993) Stichochrysis immobilis is a diatom, not a chyrsophyte. Phycologia 32:234–236CrossRefGoogle Scholar
  24. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  25. Hosoi-Tanabe S, Sako Y (2005) Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by real-time PCR assay. Mar. Biotech 7:506–514Google Scholar
  26. Jeong HJ, Jang SH, Moestrup O, Kang NS, Lee SY, Potvin E, Noh JH (2014) Ansanella granifera gen. et sp nov (Dinophyceae), a new dinoflagellate from the coastal waters of Korea. Algae 29:75–99CrossRefGoogle Scholar
  27. Kamikawa R, Nagai S, Hosoi-Tanabe S, Itakura S, Yamaguchi M, Uchida Y, Baba T, Sako Y (2007) Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments. Harmful Algae 6:413–420Google Scholar
  28. Kavanagh S, Brennan C, O'Connor L, Moran S, Salas R, Lyons J, Silke J, Maher M (2010) Real-time PCR detection of Dinophysis species in Irish coastal waters. Mar Biotech 12:534–542CrossRefGoogle Scholar
  29. Nishimura T, Hariganeya N, Tawong W, Sakanari H, Yamaguchi H, Adachi M (2016) Quantitative PCR assay for detection and enumeration of ciguatera-causing dinoflagellate Gambierdiscus spp. (Gonyaulacales) in coastal areas of Japan. Harmful Algae 52:11–22CrossRefPubMedGoogle Scholar
  30. Park TG, Park YT, Lee Y (2009) Development of a SYT09 based real-time PCR probe for detection and quantification of toxic dinoflagellate Karlodinium veneficum (Dinophyceae) in environmental samples. Phycologia 48:32–43CrossRefGoogle Scholar
  31. Penna A, Antonella P, Galluzzi L, Luca G (2013) The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species. Env Sci Pollut Res Internat 20:6851–6862CrossRefGoogle Scholar
  32. Penna A, Battocchi C, Capellacci S, Fraga S, Aligizaki K, Lemee R, Vernesi C (2014) Mitochondrial, but not rDNA, genes fail to discriminate dinoflagellate species in the genus Ostreopsis. Harmful Algae 40:40–50CrossRefGoogle Scholar
  33. Perini F, Casabianca A, Battocchi C, Accoroni S, Totti C, Penna A (2011) New approach using the real-time PCR method for estimation of the toxic marine dinoflagellate Ostreopsis cf. ovata in marine environment. PLoS One 6(3):e0017699CrossRefGoogle Scholar
  34. Place AR, Bowers HA, Bachvaroff TR, Adolf JE, Deeds JR, Sheng J (2012) Karlodinium veneficum—the little dinoflagellate with a big bite. Harmful Algae 14:179–195CrossRefGoogle Scholar
  35. Raho N, Rodriguez F, Reguera B, Marin I (2013) Are the mitochondrial cox1 and cob genes suitable markers for species of Dinophysis Ehrenberg? Harmful Algae 28:64–70CrossRefGoogle Scholar
  36. Rasmussen SA, Binzer SB, Hoeck C, Meier S, de Medeiros LS, Andersen NG, Place A, Nielsen KF, Hansen PJ, Larsen TO (2017) Karmitoxin: an amine-containing polyhydroxy-polyene toxin from the marine dinoflagellate Karlodinium armiger. J Nat Prod 80:1287–1293CrossRefPubMedGoogle Scholar
  37. Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160CrossRefPubMedGoogle Scholar
  38. Sambrook J, Fritsch EF, Maniatis T (eds) (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  39. Shao P, Chen YQ, Zhou H, Yuan J, Qu LH, Zhao D, Lin YS (2004) Genetic variability in Gymnodiniaceae ITS regions: implications for species identification and phylogenetic analysis. Mar Biol 144:215–224CrossRefGoogle Scholar
  40. Throndsen J (1978) Preservation and storage. In: Sournia A (ed) Phytoplankton manual. UNESCO, Paris, pp 69–74Google Scholar
  41. Utermöhl H (1958) Zur Vervollkomnungder quantitativen Phytoplankton-Methodik. Mitt Int Ver Ther Angew Limnol 9:1-38Google Scholar
  42. Van Wagoner RM, Deeds JR, Satake M, Ribeiro AA, Place AR, Wright JLC (2008) Isolation and characterization of karlotoxin 1, a new amphipathic toxin from Karlodinium veneficum. Tetrahedron Lett 49:6457–6461CrossRefPubMedPubMedCentralGoogle Scholar
  43. Vandersea MW, Kibler SR, Holland WC, Tester PA, Schultz TF, Faust MA, Holmes MJ, Chinain M, Litaker RW (2012) Development of semi-quantitative PCR assays for the detection and enumeration of Gambierdiscus species (Gonyaulacales, Dinophyceae). J Phycol 48:902–915CrossRefPubMedGoogle Scholar
  44. Yuan J, Li MZ, Lin SJ (2015) An improved DNA extraction method for efficient and quantitative recovery of phytoplankton diversity in natural assemblages. PLoS One 10(7):e0133060CrossRefPubMedPubMedCentralGoogle Scholar
  45. Yuan J, Mi TZ, Zhen Y, Yu ZG (2012) Development of a rapid detection and quantification method of Karenia mikimotoi by real-time quantitative PCR. Harmful Algae 17:83–91CrossRefGoogle Scholar
  46. Zhang CY, Chen GF, Zhou J, Wang YY, Lu DD (2016) Development of a quantitative PCR for detection and quantification of Prorocentrum donghaiense. J Appl Phycol 28:1683–1693CrossRefGoogle Scholar
  47. Zhang H, Litaker W, Vandersea MW, Tester P, Lin SJ (2008) Geographic distribution of Karlodinium veneficum in the US east coast as detected by ITS-ferredoxin real-time PCR assay. J Plankton Res 30:905–922CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Anna Toldrà
    • 1
  • Karl B. Andree
    • 1
  • Margarita Fernández-Tejedor
    • 1
  • Jorge Diogène
    • 1
  • Mònica Campàs
    • 1
  1. 1.IRTASant Carles de la RàpitaSpain

Personalised recommendations