Development of EvaGreen Based Real-time PCR Assay for Detection and Quantification Toxic Dinoflagellate Pfiesteria Piscicida and Field Applications

유독 와편모조류 Pfiesteria Piscicida 탐지 및 정량 분석을 위한 EvaGreen 기반 Real-time PCR기법 개발과 현장 적용

  • PARK, BUM SOO (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • JOO, JAE-HYOUNG (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • KIM, MYO-KYUNG (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • KIM, JOO-HWAN (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • KIM, JIN HO (Department of Life Science, College of Natural Sciences, Hanyang University) ;
  • BAEK, SEUNG HO (South Sea Institute, KIOST) ;
  • HAN, MYUNG-SOO (Department of Life Science, College of Natural Sciences, Hanyang University)
  • Received : 2016.08.16
  • Accepted : 2017.02.22
  • Published : 2017.02.28


Pfiesteria piscicida is one of heterotrophic dinoflagellate having toxic metaboliges, and it is difficult to detect and quantify this dinoflagellate via light microscope due to small size and morphological similarity with Pfiesteria-like dinoflagellate (PLD) species. Alternatively, we developed quantitative real-time PCR assay based on EvaGreen and determined field accessibility throughout the investigation of distribution in the entire Korean coastal waters and population dynamics in Shihwa Lake. The P. piscicida-specific primers based on internal transcribed spacer 1 (ITS 1) were designed and the specificity of primers was confirmed by PCR with other genomic DNAs which have genetic similarity with target species. Through real-time PCR assay, a standard curve which had a significant linear correlation between log cell number and $C_T$ value ($r^2{\geq}0.999$) and one informative melting peak ($88^{\circ}C$) were obtained. These results implies that developed real-time PCR can accurately detect and quantify P. piscicida. Throughout the field applications of real-time PCR assay, P. piscicida was distributed in western (Mokpo and Kimje) and easthern (Gangneng) Korean coastal water even though light microscopy failed to identify P. piscicida. In the investigation of population dynamics in Shihwa Lake, the density of P. piscicida was peaked in June, July and August 2007 at St. 1 where salinity (${\leq}15psu$) was lower than the other 2 sites. In this study, we successed to develop EvaGreen bassed real-time PCR for detection and quantification of P. piscicida in fields, so this developed assay will be useful for various ecological studies in the future.


Supported by : 해양수산부


  1. Arikawa, E., Y. Sun, J. Wang, Q. Zhou, B. Ning, S.L. Dial, L. Guo and J. Yang, 2008. Cross-platform comparison of $SYBR^{(R)}$ Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC) study. BMC Genomics, 9: 328. doi: 10.1186/1471-2164-9-328.
  2. Audemard, C., K.S. Reece and E.M. Burreson, 2004. Real-time PCR for detection and quantification of the protistan parasite Perkinsus marinus in environmental waters. Appl. Environ. Microbiol., 70: 6611-6618.
  3. Audemard, C., L.M. Ragone Calvo, K.T. Paynter, K.S. Reece and E.M. Burreson, 2006. Real-time PCR investigation of parasite ecology: In situ determination of oyster parasite Perkinsus marinus transmission dynamics in lower Chesapeake Bay. Parasitology, 132: 827-842.
  4. Bartlett, G.R., 1958. Phosphorus assay in column chromatography. J. Biological Chem., 234: 466-468.
  5. Baek, S.H., K. You, B.S. Park and, Han, M-.S. Han, 2010. The seasonal variation of microbial community in the eutrophic brackish water of Lake Shihwa. Korean J. Limnol. 43:55-68.
  6. Bowers, H.A., T. Tengs, H.B. Glasgow, J.M. Burkholder, P.A. Rublee and D.W. Oldach, 2000. Development of Real-Time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellate. Appl. Environ. Microbiol., 66: 4641-4648.
  7. Burkholder, J.M. and H.B. Glasgow, 1997. Pfiesteria piscicida and other Pfiesteria-like dinoflagellates: Behavior, impacts, and environmental controls. Limnology, 42: 1052-1075.
  8. Burkholder, J.M., A.S. Gordon, P.D. Moeller, J.M. Law, K.J. Coyne, A.J. Lewitus, J.S. Ramsdell, H.G. Marshall, N.J. Deamer, S.C., Cary, J.W. Kempton, S.L. Morton and P.A. Rublee, 2005. Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains. Proc. Natl. Am. Soc. U.S.A., 102: 3471-3476.
  9. Burkholder, J.M., E.J. Noga, C.H. Hobbs and H.B. Glasgow, 1992. New 'Phantom' dinoflagellate is the causative agent of major estuarine fish kills. Nature, 358: 407-410.
  10. Burkholder, J.M., H.G. Marshall, H.B. Glasgow, D.W. Seaborn and N.J. Deamer-Melia, 2001. The Standardized Fish Bioassay Procedure for Detecting and Culturing Actively Toxic Pfiesteria, Used by Two Reference Laboratories for Atlantic and Gulf Coast States. Environ. Health Persp., 109: 745-756.
  11. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol., 25: 169-193.
  12. Bustin, S.A., 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol., 29: 23-39.
  13. Caron, D.A., 1983. Technique for Enumeration of Heterotrophic and Phototrophic Nanoplankton, Using Epifluorescence Microscopy, and Comparison with Other Procedures. Appl. Environ. Microbiol., 46: 491-498.
  14. Coyne, K.J., S.M. Handy, E. Demir, E.B. Whereat, D.A. Hutchins, K.J. Portune, M.A. Doblin and S.C. Cary, 2005. Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnol. Oceanogra., 3: 381-391.
  15. Faveri, J., R.M. Smolowitz and S.B. Robert, 2009. Development and validation of a real-time PCR assay for the detection and quantification of Perkinsus marinus in the Eastern oyster, Crassostrea virginica. J. Shellfish Res., 28: 459-464.
  16. Galluzzi, L., A. Penna, E. Bertozzini, M. Vila, E. Garces and M. Magnani, 2004. Development of a Real-Time PCR Assay for Rapid Detection and Quantification of Alexandrium minutum (a Dinoflagellate). Appl. Environ. Microbiol., 70: 1199-1206.
  17. Glasgow, H.B., J. M. Burkholder, J. M. Morton and J. Springer, 2001. A second species of ichthyotoxic Pfiesteria (Dinamoebales, Dinophyceae). Phycologia, 40: 234-245.
  18. Grattan, L.M., D. Oldach, T.M. Perl, M.H. Lowitt, D.L. Matuszak, C. Dickson, C. Parrott, R.C. Shoemaker, C.L. Kauffman, M.P. Wasserman, R. Hebel, P. Charache and G. Morris, 1998. Learning and memory difficulties after environmental exposure to waterways containing toxin-producing Pfiesteria or Pfiesteria-like dinoflagellates. Lancet, 352: 532-539.
  19. Gray, M.B. B. Wawrik, J. Paul and E. Casper, 2003. Molecular Detection and Quantitation of the Red Tide Dinoflagellate Karenia brevis in the Marine Environment. Appl. Environ. Microbiol., 69: 5726-5730.
  20. Grkstatten, J.H., M.O. Allum, S.E. Dominguez and M.R. Crouse, 1978. A Survay of Phosphous and Nitrogen Levels in Treated Municipal Wastewater. Jour. WPCF., 50: 718-722.
  21. Guay, J.M., A. Huot, S. Gagnon, A. Tremblay and R.C. Levesque, 1992. Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma-gondii-primary and secondary structure of the 5s gene. Gene, 114: 165-171.
  22. Harder, T., C.K.S Lau., S. Dobretsov, T.K. Fang and P.Y. Qian, 2003. A distinctive epibiotic bacterial community on the soft Dendronephthya sp. and antibacterial activity of coral tissue extracts suggest a chemical mechanism against bacterialepibiosis. FEMS Microb. Ecol., 43: 337-347.
  23. Haywood, A.J., C.A. Scholin, R. Marin III, K.A. Steidinger, C. Heil and J. Ray, 2007. Molecular detection of the brevetoxin-producing dinoflagellate Karenia brevis and closely related species using rRNA-targeted probes and a semiautomated sandwich hybridization assay. J. Phycol., 43: 1271-1286.
  24. Jeffrey, S.W. and G.F. Humphrey, 1975. New spectrophotometic equations for determining chlorophylls a, b, c, and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol., 167: 191-194.
  25. Jeong, H.J., J.H. Ha, J.Y. Park, J.H. Kim, N.S. Kang, S.H. Kim, J.S. Kim, Y.D. Yoo and W.H. Yih, 2006. Distribution of the heterotrophic dinoflagellate Pfiesteria piscicida in Korean waters and its consumption of mixotrophic dinoflagellates, raphidophytes and fish blood cells. Aquat. Microbiol. Ecol., 44: 263-278.
  26. Jeong, H.J., J.S. Kim, J.Y. Song, J.H. Kim, T.H. Kim, S.K. Kim and N.S. Kang, 2007. Feeding by protists and copepods on the heterotrophic dinoflagellates Pfiesteria piscicida, Stoeckeria algicida, and Luciella masanensis. Mar. Ecol. Prog. Ser., 349: 199-211.
  27. Kamphake, L.J., S.A. Hannah and J.M. Cohen, 1967. Automated anlaysis for nitrate by hydrazine reduction. Water Res., 1: 205-216.
  28. Kane, A.K., D. Oldach and R. Reimschuessel, 1998. Fish Lesions in The Chesapeake Bay: Pfiesteria-like Dinoflagellates and Other Etiologies. Md. Med. J., 47: 106-112.
  29. Kreuzer, K.A., U. Lass, O. Landt, A. Nitsche, J. Laser, H. Ellerbrok, G. Pauli, D. Huhn and C.A. Schmidt, 1999. Highly Sensitive and Specific Fluorescence Reverse Transcription-PCR Assay for the Pseudogene-free Detection of ${\beta}$-Actin Transcripts as Quantitative Reference. Clin. Chem., 45: 297-300.
  30. Le Blancq, S.M., N.V. Khramtsov, F. Zamani, S.J. Upton and T.W. Wu, 1997. Ribosomal RNA gene organization in Cryptosporidium pavum. Mol. Biochem. Parasitol., 90: 463-478.
  31. Lewitus, A.J., R.V. Jesien, T.M. Kana, J.M. Burkholder and E. May, 1995. Discovery of the "Phantom" Dinoflagellate in Chesapeak bay. Estuaries, 18: 373-378.
  32. Litaker, R. W., M. W. Vandersea, S. R. Kibler, R. Steven, K. S. Reece, S. Kimberly, N. A. Stokes, F. M. Lutzoni, B. A. Yonish, M. A. West, M. N. D. Black, and P. A. Tester. 2007. Recognizing dinoflagellate species using ITS rDNA sequences. J. Phycol. 43:344-355.
  33. Litaker, R.W., M.W. Vandersea, S.R. Kibler, V.J. Madden, E.J. Noga and P.A. Tester, 2002. Life cycle of the heterotrophic dinoflagellate Pfiesteria piscicida (dinophyceae). J. Phycol., 38: 442-463.
  34. Lowry, O.H. and J.A. Lopez, 1945. The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem., 162: 421-428.
  35. Mao, F., W.Y. Leung and X. Xin, 2007. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol., 7: 76 doi: 10.1186/1472-6750-7-76.
  36. Marshall, H.G., A.S. Gordon, D.W. Seaborn, B. Dyer, W.M. Dunstan and A.M. Seaborn, 2000. Comparative culture and toxicity studies between the toxic dinoflagellate Pfiesteria piscicida and a morphologically similar cryptoperidiniopsoid dinoflagellate. J. Exp. Mar. Biol. Ecol., 255: 51-74.
  37. Oldach, D.W., C.F. Delwiche, K.S. Jakobsen, T. Tengs, E.G. Brown, J.W. Kempton, E.F. Schaefer, H. Bowers, K. Steidinger, H.B. Glasgow, J.M. Burkholder and P.A. Rublee, 2000. Heteroduplex mobility assay guided sequence discovery: elucidation of the small subunit (18S) rDNA sequence of Pfiesteria piscicida from complex algal culture and environmental sample DNA pools. Proc. Natl. Ac. Sci. U.S.A., 97: 4303-4308.
  38. Park, B.S., P. Wang, J.H. Kim, J-H. Kim, C.J. Gobler and M-S. Han, 2014. Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal water via use of quantitative PCR assays. Harmful algae, 37: 133-141.
  39. Park, B.S., S.H. Baek, J-S. Ki, R.A. Cattolico and M-S. Han, 2012. Assessment of EvaGreen-based quantitative real-time PCR assay for enumeration of the microalgae Heterosigma and Chattonella (Raphidophyceae). J. Appl. Phycol., 24: 1555-1567.
  40. Park, T.G., F. Miguel, J.S. Bolch and G.M. Hallegraeff, 2007. Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate Cryptoperidiniopsis brodyi (Dinophyceae) in Environmental Samples. Appl. Environ. Microbiol., 73: 2552-2560.
  41. Rebricov, C.V. and D.Y. Trofimov, 2006. Real-Time PCR: a review of approaches to data analysis. Appl. Biochem. Microbiol., 42: 455-463.
  42. Saito, K., T. Drgon, J.A.F. Robledo, D.N. Krupatkina and G.R. Vasta, 2002. Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the non-transcribed spacer. Appl. Environ. Microbiol., 68: 5394-5407.
  43. Steidinger, K., J. Landsberg, R.W. Richardson, B. Blakesley, P. Scott, P. Tester, T. Tengs, P. Mason, S. Morton, D. Seaborn, W. Litaker, K. Reece, D. Oldach, L. Haas and G. Vasta, 2001. Classification and Identification of Pfiesteria and Pfiesteria-Like Species. Environ. Health Persp., 109: 661-665.
  44. Thompson, J.D., T.J. Gibson, F. Plewnaik, F. Jeanmougin and D.G. Higgins, 1997. The CLUSTALX windows interface: flexible strategies for multiple sequence alignment aided by quality tools. Nucleic Acids Res., 25: 4876-4882.
  45. Vadopalas, B., J.V. Bouma, C.R. Jackels and C.S. Friedman, 2006. Application of real-time PCR for simultaneous identification and quantification of larval abalone. J. Exp. Mar. Biol. Ecol., 334: 219-228.