ALGAE

  • 제32권3호
  • /
  • Pages.189-197
  • /
  • 2017
  • /
  • 1226-2617(pISSN)
  • /
  • 2093-0860(eISSN)
한국조류학회(藻類) (The Korean Society of Phycology)
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An advanced tool, droplet digital PCR (ddPCR), for absolute quantification of the red-tide dinoflagellate, Cochlodinium polykrikoides Margalef (Dinophyceae)

  • Lee, Hyun-Gwan (Department of Oceanography, Chonnam National University) ;
  • Kim, Hye Mi (Department of Oceanography, Chonnam National University) ;
  • Min, Juhee (Department of Oceanography, Chonnam National University) ;
  • Kim, Keunyong (Department of Oceanography, Chonnam National University) ;
  • Park, Myung Gil (Department of Oceanography, Chonnam National University) ;
  • Jeong, Hae Jin (School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University) ;
  • Kim, Kwang Young (Department of Oceanography, Chonnam National University)
  • 투고 : 2017.04.03
  • 심사 : 2017.09.10
  • 발행 : 2017.09.30
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초록

To quantify the abundance of the harmful dinoflagellate Cochlodinium polykrikoides in natural seawaters, we developed the innovative procedure using a droplet digital PCR (ddPCR) with C. polykrikoides-specific primers targeting the internal transcription sequence (ITS). The abundance of C. polykrikoides was estimated by the specific copy number of target ITS DNA segments per cell in cultures and natural water samples. The copy number per C. polykrikoides cell as acquired by ddPCR was $157{\pm}16$, which was evaluated against known cell numbers through a simplified protocol preparing DNAs. The abundances of C. polykrikoides in the waters of different locations estimated by ddPCR agreed with the number of cells visually counted under a microscope. This protocol was used to measure the abundance of C. polykrikoides close to and further off the southern coast of Korea in August of 2016 and 2017. The practical application showed that this method can reduce time for analysis and increase accuracy.

키워드

참고문헌

  1. Bereiter-Hahn, J. 1990. Behavior of mitochondria in the living cell. Int. Rev. Cytol. 122:1-63.
  2. Buchheim, M. A., Keller, A., Koetschan, C., Forster, F., Merget, B. & Wolf, M. 2011. Internal transcribed spacer 2 (nu ITS2 rRNA) sequence-structure phylogenetics: towards an automated reconstruction of the green algal tree of life. PLoS One 6:e16931. https://doi.org/10.1371/journal.pone.0016931
  3. Cho, E. S., Kim, G. Y., Choi, B. D., Rhodes, L. L., Kim, T. J., Kim, G. H. & Lee, J. D. 2001. A comparative study of the harmful dinoflagellates Cochlodinium polykrikoides and Gyrodinium impudicum using transmission electron microscopy, fatty acid composition, carotenoid content, DNA quantification and gene sequences. Bot. Mar. 44:57-66.
  4. Coleman, A. W. & Mai, J. C. 1997. Ribosomal DNA ITS-1 and ITS-2 sequence comparisons as a tool for predicting genetic relatedness. J. Mol. Evol. 45:168-177. https://doi.org/10.1007/PL00006217
  5. Coyne, K. J., Handy, S. M., Demir, E., Whereat, E. B., Hutchins, D. A., Portune, K. J., Doblin, M. A. & 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. https://doi.org/10.4319/lom.2005.3.381
  6. Devonshire, A. S., Honeyborne, I., Gutteridge, A., Whale, A. S., Nixon, G., Wilson, P., Jones, G., McHugh, T. D., Foy, C. A. & Huggett, J. F. 2015. Highly reproducible absolute quantification of Mycobacterium tuberculosis complex by digital PCR. Anal. Chem. 87:3706-3713. https://doi.org/10.1021/ac5041617
  7. Ellison, S. L. R., Emslie, K. R. & Kassir, Z. 2011. A standard additions method reduces inhibitor-induced bias in quantitative real-time PCR. Anal. Bioanal. Chem. 401:3221-3227. https://doi.org/10.1007/s00216-011-5460-y
  8. Flekna, G., Schneeweiss, W., Smulders, F. J. M., Wagner, M. & Hein, I. 2007. Real-time PCR method with statistical analysis to compare the potential of DNA isolation methods to remove PCR inhibitors from samples for diagnostic PCR. Mol. Cell. Probes 21:282-287. https://doi.org/10.1016/j.mcp.2007.02.001
  9. Hong, H. -H., Lee, H. -G., Jo, J., Kim, H. M., Kim, S. -M., Park, J. H., Jeon, C. B., Kang, H. -S., Park, M. G., Park, C. & Kim, K. Y. 2016. The exceptionally large genome of the harmful red tide dinoflagellate Cochlodinium polykrikoides Margalef (Dinophyceae): determination by flow cytometry. Algae 31:373-378. https://doi.org/10.4490/algae.2016.31.12.6
  10. Iwataki, M., Hansen, G., Moestrup, O. & Matsuoka, K. 2010. Ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with reference to the apical groove and flagellar apparatus. J. Eukaryot. Microbiol. 57:308-321. https://doi.org/10.1111/j.1550-7408.2010.00491.x
  11. Jeffrey, S. W. & Vesk, M. 1997. Introduction to marine phytoplankton and their pigment signatures. In Jeffrey, S. W., Mantoura, R. F. C. & Wright, S. W. (Eds.) Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods. Vol. 10. Monographs on Oceanographic Methodology. UNESCO Publishing, Paris, pp. 37-84.
  12. Jeong, H. J., Lee, K., Yoo, Y. D., Kim, J. -M., Kim, T. H., Kim, M., Kim, J. -H. & Kim, K. Y. 2016. Reduction in $CO_2$ uptake rates of red tide dinoflagellates due to mixotrophy. Algae 31:351-362. https://doi.org/10.4490/algae.2016.31.11.17
  13. Kim, C. -J., Kim, H. -G., Kim, C. -H. & Oh, H. -M. 2007. Life cycle of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters. Harmful Algae 6:104-111. https://doi.org/10.1016/j.hal.2006.07.004
  14. Kudela, R. M. & Gobler, C. J. 2012. Harmful dinoflagellate blooms caused by Cochlodinium sp.: global expansion and ecological strategies facilitating bloom formation. Harmful Algae 14:71-86. https://doi.org/10.1016/j.hal.2011.10.015
  15. Lee, C. -K., Park, T. -G., Park, Y. -T. & Lim, W. -A. 2013. Monitoring and trends in harmful algal blooms and red tides in Korean coastal waters, with emphasis on Cochlodinium polykrikoides. Harmful Algae 30(Suppl. 1):S3-S14. https://doi.org/10.1016/j.hal.2013.10.002
  16. Lee, S. Y., Jeong, H. J., Seong, K. A., Lim, A. S., Kim, J. H., Lee, K. H., Lee, M. J. & Jang, S. H. 2017. Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods. Harmful Algae 63:23-31. https://doi.org/10.1016/j.hal.2017.01.006
  17. Marcoval, M. A., Pan, J., Tang, Y. & Gobler, C. J. 2013. The ability of the branchiopod, Artemia salina, to graze upon harmful algal blooms caused by Alexandrium fundyense, Aureococcus anophagefferens, and Cochlodinium polykrikoides. Estuar. Coast. Shelf Sci. 131:235-244. https://doi.org/10.1016/j.ecss.2013.05.034
  18. Matsuoka, K., Iwataki, M. & Kawami, H. 2008. Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae). Harmful Algae 7:261-270. https://doi.org/10.1016/j.hal.2007.12.002
  19. McKibben, S. M., Watkins-Brandt, K. S., Wood, A. M., Hunter, M., Forster, Z., Hopkins, A., Du, X., Eberhart, B. -T., Peterson, W. T. & White, A. E. 2015. Monitoring Oregon Coastal Harmful Algae: observations and implications of a harmful algal bloom-monitoring project. Harmful Algae 50:32-44. https://doi.org/10.1016/j.hal.2015.10.004
  20. Park, B. S., Wang, P., Kim, J. H., Kim, J. -H., Gobler, C. J. & Han, M. -S. 2014. Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays. Harmful Algae 37:133-141. https://doi.org/10.1016/j.hal.2014.04.019
  21. Park, J., Jeong, H. J., Yoon, E. Y. & Moon, S. J. 2016. Easy and rapid quantification of lipid contents of marine dinoflagellates using the sulpho-phospho-vanillin method. Algae 31:391-401. https://doi.org/10.4490/algae.2016.31.12.7
  22. Park, T. G., Lim, W. A., Park, Y. T., Lee, C. K. & Jeong, H. J. 2013. Economic impact, management and mitigation of red tides in Korea. Harmful Algae 30(Suppl. 1):S131-S143. https://doi.org/10.1016/j.hal.2013.10.012
  23. Richlen, M. L., Morton, S. L., Jamali, E. A., Rajan, A. & Anderson, D. M. 2010. The catastrophic 2008-2009 red tide in the Arabian gulf region, with observations on the identification and phylogeny of the fish-killing dinoflagellate Cochlodinium polykrikoides. Harmful Algae 9:163-172. https://doi.org/10.1016/j.hal.2009.08.013
  24. Rountos, K. J., Tang, Y. -Z., Cerrato, R. M., Gobler, C. J. & Pikitch, E. K. 2014. Toxicity of the harmful dinoflagellate Cochlodinium polykrikoides to early life stages of three estuarine forage fish. Mar. Ecol. Prog. Ser. 505:81-94. https://doi.org/10.3354/meps10793
  25. Sanders, R., Huggett, J. F., Bushell, C. A., Cowen, S., Scott, D. J. & Foy, C. A. 2011. Evaluation of digital PCR for absolute DNA quantification. Anal. Chem. 83:6474-6484. https://doi.org/10.1021/ac103230c
  26. Sato, N., Terasawa, K., Miyajima, K. & Kabeya, Y. 2003. Organization, developmental dynamics, and evolution of plastid nucleoids. Int. Rev. Cytol. 232:217-262.
  27. Scollo, F., Egea, L. A., Gentile, A., La Malfa, S., Dorado, G. & Hernandez, P. 2016. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): comparison of isolation and amplification methodologies. Food Chem. 213:388-394. https://doi.org/10.1016/j.foodchem.2016.06.086
  28. Sellner, K. G., Doucette, G. J. & Kirkpatrick, G. J. 2003. Harmful algal blooms: causes, impacts and detection. J. Ind. Microbiol. Biotechnol. 30:383-406. https://doi.org/10.1007/s10295-003-0074-9
  29. Shahraki, J., Motallebi, A., Barekati, I., Seydi, E. & Pourahmad, J. 2014. Comparison of cellular and molecular cytotoxic mechanisms of Cochlodinium polykrikoides in isolated trout and rat hepatocytes. Toxicol. Environ. Chem. 96:917-930. https://doi.org/10.1080/02772248.2014.980132
  30. Shahraki, J., Motallebi, A. & Pourahmad, J. 2013. Oxidative mechanisms of fish hepatocyte toxicity by the harmful dinoflagellate Cochlodinium polykrikoides. Mar. Environ. Res. 87-88:52-60. https://doi.org/10.1016/j.marenvres.2013.03.004
  31. Wilson, I. G. 1997. Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63:3741-3751.

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