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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

  • 박범수 (한양대학교 생명과학과) ;
  • 주재형 (한양대학교 생명과학과) ;
  • 김묘경 (한양대학교 생명과학과) ;
  • 김주환 (한양대학교 생명과학과) ;
  • 김진호 (한양대학교 생명과학과) ;
  • 백승호 (한국해양과학기술원 남해특성연구센터) ;
  • 한명수 (한양대학교 생명과학과)
  • 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

초록

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.

키워드

Pfiesteria piscicida;quantitative real-time PCR;field application

과제정보

연구 과제 주관 기관 : 해양수산부

참고문헌

  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. https://doi.org/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. https://doi.org/10.1128/AEM.70.11.6611-6618.2004
  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. https://doi.org/10.1017/S0031182006009851
  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. https://doi.org/10.1128/AEM.66.11.4641-4648.2000
  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. https://doi.org/10.1073/pnas.0500168102
  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. https://doi.org/10.1038/358407a0
  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. https://doi.org/10.1289/ehp.01109s5745
  11. Bustin, S.A., 2000. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol., 25: 169-193. https://doi.org/10.1677/jme.0.0250169
  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. https://doi.org/10.1677/jme.0.0290023
  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. https://doi.org/10.4319/lom.2005.3.381
  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. https://doi.org/10.2983/035.028.0306
  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. https://doi.org/10.1128/AEM.70.2.1199-1206.2004
  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. https://doi.org/10.2216/i0031-8884-40-3-234.1
  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. https://doi.org/10.1016/S0140-6736(98)02132-1
  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. https://doi.org/10.1128/AEM.69.9.5726-5730.2003
  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. https://doi.org/10.1016/0378-1119(92)90570-F
  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. https://doi.org/10.1111/j.1574-6941.2003.tb01074.x
  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. https://doi.org/10.1111/j.1529-8817.2007.00407.x
  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. https://doi.org/10.3354/ame044263
  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. https://doi.org/10.3354/meps07094
  27. Kamphake, L.J., S.A. Hannah and J.M. Cohen, 1967. Automated anlaysis for nitrate by hydrazine reduction. Water Res., 1: 205-216. https://doi.org/10.1016/0043-1354(67)90011-5
  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. https://doi.org/10.1016/S0166-6851(97)00181-3
  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. https://doi.org/10.2307/1352319
  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. https://doi.org/10.1111/j.1529-8817.2007.00320.x
  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. https://doi.org/10.1046/j.1529-8817.2002.t01-1-01242.x
  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. https://doi.org/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. https://doi.org/10.1016/S0022-0981(00)00288-4
  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. https://doi.org/10.1073/pnas.97.8.4303
  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. https://doi.org/10.1016/j.hal.2014.04.019
  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. https://doi.org/10.1007/s10811-012-9816-2
  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. https://doi.org/10.1128/AEM.02389-06
  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. https://doi.org/10.1134/S0003683806050024
  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. https://doi.org/10.1128/AEM.68.11.5394-5407.2002
  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. https://doi.org/10.1289/ehp.01109s5661
  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. https://doi.org/10.1093/nar/25.24.4876
  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. https://doi.org/10.1016/j.jembe.2006.02.005