The Korean Journal of Mycology (한국균학회지)

The Korean Society of Mycology (한국균학회)
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Species-specific Marker of Phytophthora pinifolia for Plant Quarantine in Korea

국내 식물검역대상 Phytophthora pinifolia의 PCR 검출을 위한 종 특이적 마커 개발

  • Kim, Narae (Department of Applied Biology, Chungnam National University) ;
  • Choi, You Ri (Department of Applied Biology, Chungnam National University) ;
  • Seo, Mun Won (Department of Applied Biology, Chungnam National University) ;
  • Song, Jeong Young (Department of Applied Biology, Chungnam National University) ;
  • Kim, Hong Gi (Department of Applied Biology, Chungnam National University)
  • 김나래 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 최유리 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 서문원 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 송정영 (충남대학교 농업생명과학대학 응용생물학과) ;
  • 김홍기 (충남대학교 농업생명과학대학 응용생물학과)
  • Received : 2016.04.19
  • Accepted : 2016.06.23
  • Published : 2016.06.30
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Abstract

To establish a rapid and accurate detection of Phytophthora pinifolia, which is a quarantine pathogenic fungus in Korea, a species-specific primer was developed based on the ras-related protein (Ypt1) gene. Species-specific primer based on the DNA sequences of Ypt1 gene amplified 193 bp polymerase chain reaction (PCR) product for P. pinifolia. The primer pair yielded the predicted PCR product size exactly in testing with target pathogen DNAs, but not from the other 10 species of Phytophthora and 14 species of other phytopathogenic fungi. The primer pair also showed only the species-specific amplification curve on realtime PCR on target pathogen DNA. The detection sensitivity of real time PCR using species-specific primer pair was 10 to 100 times higher than conventional PCR, with 1 to $10pg/{\mu}L$.

본 연구는 국내 주요 식물검역관리 대상 Phytophthora pinifolia를 대상으로 신속하고 정확한 병원균 종동정 및 검출을 위해 Ypt1 유전자 염기서열을 활용하여 제작된 종 특이적 분석용 분자마커와 다양한 PCR 기법을 활용하여 병원균들에 대한 다양한 검출기술의 표준화 및 최적 검출 시스템 구축을 통하여 실제 검역검역 현장에서 활용 가능한 병원균 존재여부의 신속, 정확한 판별기법을 개발하고자 수행하였다. Ypt1 영역의 염기서열을 기초로 선발된 종 특이적 primer는 1~10 pg의 검출민감도를 가지며 193 bp의 종 특이적 PCR 증폭산물을 형성시켰다. 또한 선발된 종 특이적 primer의 종 특이성을 확인하기 위하여 국내 식물검역대상에 포함된 Phytophthora속 종들과 주요 식물병원균들을 대상으로 conventional PCR과 real-time PCR을 수행한 결과 목표로 한 P. pinifolia DNA에서만 특이적 PCR 증폭산물을 확인할 수 있었다. 선발된 종 특이적 primer에 대한 PCR의 검출 민감도를 확인했을 때 conventional PCR의 검출민감도는 1~10 pg이었다.

Keywords

References

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