The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY (한국해양학회지:바다)

The Korean Society of Oceanography (한국해양학회)
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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
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Abstract

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.

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.

Keywords

References

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