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

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

  • 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)
  • 투고 : 2016.08.16
  • 심사 : 2017.02.22
  • 발행 : 2017.02.28


Pfiesteria piscicida는 유독 종속영양 와편모조류로서, 크기가 작고 형태학적으로 유사한 Pfiesteria-like dinoflagellate (PLD) 종들로 인해, 광학 현미경 관찰만으로 정확하게 동정하는 것이 불가능하다. 따라서, 본 연구에서는 이러한 한계점을 극복하기 위해 EvaGreen 기반의 정량적 real-time PCR기법을 개발하였으며, 한국 근해에서 P. piscicida의 분포와 시화호에서 개체군 변동 조사를 통해 현장에서 유용성을 검증하였다. 이를 위해, internal transcribed spacer 1 (ITS 1) 영역을 대상으로 종 특이적 프라이머를 제작하였으며, P. piscicida와 진화적 유연관계에 있는 다양한 미세조류에 대해 PCR을 수행하여 프라이머의 특이성을 검증하였다. 개발된 프라이머를 real-time PCR 기법에 적용한 결과, P. piscicida의 세포수와 $C_T$값 간의 유의한 표준 곡선($r^2{\geq}0.999$)과 하나의 융해곡선 피크($88^{\circ}C$)가 관찰되었다. 이는 본 연구에서 개발된 기법이 대상생물인 P. piscicida를 정확하게 정성 및 정량분석이 가능함을 의미한다. 개발된 real-time PCR 기법의 현장적용 결과, 광학 현미경상에서는 탐지할 수 없었던 P. piscicida를 서해(김제, 목포)와 동해(강릉) 시료에서 검출하였다. 또한, 시화호 시료를 이용한 P. piscicida개체군 동태 조사에서 다른 정점에 비해 염분도가 상대적으로 낮았던(${\leq}15psu$), St. 1에서 2007년 6, 7, 8월에 세포밀도의 피크가 관찰되었다. 본 연구에서 개발된 EvaGreen 기반 real-time PCR 기법은 현장에서 P. piscicida를 탐지 및 정량 분석 하는데 성공하였으며, 이는 향후 이들 종에 대한 다양한 생태학적 연구에 활용될 것으로 사료된다.


연구 과제 주관 기관 : 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.