미생물학회지 (Korean Journal of Microbiology)

  • 제47권2호
  • /
  • Pages.103-109
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  • 2011
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  • 0440-2413(pISSN)
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  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

Loop-Mediated Isothermal Amplification (LAMP)법을 이용한 Pectobacterium carotovorum subsp. carotovorum의 신속 진단법 개발

Development of a Rapid Detection Method for Pectobacterium carotovorum subsp. carotovorum Using the Loop-Mediated Isothermal Amplification (LAMP)

  • 김정구 (국립농업과학원 유전자분석개발과) ;
  • 노지나 (경기대학교 일반대학원 생명과학과) ;
  • 박동석 (국립농업과학원 유전자분석개발과) ;
  • 윤병수 (경기대학교 일반대학원 생명과학과)
  • Kim, Jeong-Gu (Genomics Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • No, Ji-Na (Department of Life Science, College of Natural Science, Kyonggi University) ;
  • Park, Dong-Suk (Genomics Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • Yoon, Byoung-Su (Department of Life Science, College of Natural Science, Kyonggi University)
  • 투고 : 2011.04.28
  • 심사 : 2011.06.15
  • 발행 : 2011.06.30
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초록

Pectobacterium carotovorum subsp. carotovorum (PCC)는 세균성 무름병균으로 주로 감자, 양배추 등의 식물에서 질병을 일으킨다. 본 연구에서는 현장에서 신속하게 진단하기 위해 loop-mediated isothermal amplification법을 이용하여 1시간 내에 등온에서 검출 가능한 진단법을 개발하였으며, 이를 PCC-LAMP법이라 명명하였다. PCC의 lytic murein transglycolase 유전자를 특이적으로 증폭시키는 4개의 프라이머를 제작하였으며 최적 온도가 $61^{\circ}C$임을 확인하고 최적 조건을 확립하였다. 최적 조건을 바탕으로 4개의 프라이머가 $1{\times}10^3$ copies까지 검출하는 민감성을 확인할 수 있었다. 본 연구에서 개발된 PCC-LAMP법은 특이성 검사를 통해 PCC만이 특이적으로 검출됨을 확인하였으며, 이는 실제 시료에서도 적용 가능함을 확인하였다. PCC-LAMP법을 통하여 PCC를 신속하고 정확하게 검출함으로써 현장에서 유용하게 적용될 수 있을 것으로 사료된다.

Pectobacterium carotovorum subsp. carotovorum is the causative agent of soft rot in crops such as potato and cabbages. Loop-mediated isothermal amplification (LAMP) is a simple DNA amplification method, as well as isothermal PCR technique. In this study, a new method for the rapid detection of Pectobacterium carotovorum subsp. carotovorum was developed using LAMP that named PCC-LAMP. Based on lytic murein transglycolase gene of Pectobacterium carotovorum subsp. carotovorum, a set of four primers for LAMP was designed. The optimal PCC-LAMP reaction temperature was established at $61^{\circ}C$. Under standard conditions, PCC-LAMP amplified $1{\times}10^3$ copies of clone PCC-pBX437 per reaction. Further, this method can also assay directly by SYBR Green I without electrophoresis. Amplification was not detected for five other bacterial species. In conclusion, PCC-LAMP may be a useful method for the detection Pectobacterium carotovorum subsp. carotovorum in the field.

키워드

참고문헌

  1. Aoi, Y., M. Hosogai, and S. Tsuneda. 2006. Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria. J. Biotechnol. 125, 484-491. https://doi.org/10.1016/j.jbiotec.2006.04.007
  2. Blomstrom, A.L., M. Hakhverdyan, S.M. Reid, J.P. Dukes, D.P. King, S. Belak, and M. Berg. 2008. A one-step reverse transcriptase loop-mediated isothermal amplification assay for simple and rapid detection of swine vesicular disease virus. J. Virol. Methods 147, 188-193. https://doi.org/10.1016/j.jviromet.2007.08.023
  3. Kang, H.W., S.J. Go, and S.W. Kwon. 1998. PCR specific detection of Erwinia carotovora subsp. carotovora by DNA probe selected from PCR polymorphic bands. Plant Pathol. J. 14, 164-170.
  4. Kiatpathomchai, W., W. Jareonram, S. Jitrapakdee, and T.W. Flegel. 2007. Rapid and sensitive detection of Taura syndrome virus by reverse transcription loop-mediated isothermal amplification. J. Virol. Methods 146, 125-128. https://doi.org/10.1016/j.jviromet.2007.06.007
  5. Kim, Y.C., D.U. Song, B.H. Cho, and K.C. Kim. 1993. Cloning of the DNA fragment involved in the pathogenicity of Erwinia carotovora subsp. carotovora, plant soft rot bacterium. Plant Pathol. J. 9, 213-217.
  6. Nagamine, K., T. Hase, and T. Notomi. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol. Cell. Probes 16, 223-229. https://doi.org/10.1006/mcpr.2002.0415
  7. No, J.N., M.S. Yoo, D.S. Park, J.G. Kim, and B.S. Yoon. 2009. Development of a new PCR method for detection of Pectobacterium carotovorum. Kor. J. Microbiol. 45, 306-311.
  8. Ohori, A., S. Endo, A. Sano, K. Yokoyama, K. Yarita, M. Yamaguchi, K. Kamei, M. Miyaji, and K. Nishimura. 2006. Rapid identification of Ochroconis gallopava by a loop-mediated isothermal amplification (LAMP) method. Vet. Microbiol. 114, 359-365. https://doi.org/10.1016/j.vetmic.2005.11.064
  9. Roh, E., S. Lee, D. Fa, J. Choi, E. Moon, and S. Heu. 2009. Diverse antibacterial activity of Pectobacterium carotovorum subsp. carotovorum isolated in Korea. J. Microbiol. Biotechnol. 19, 42-50.
  10. Tsugunori, N., H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, and T. Hase. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, e63. https://doi.org/10.1093/nar/28.12.e63
  11. Tsugunori, N. 2007. Loop-mediated isothermal amplification. Nippon Rincho. 65, 957-961.
  12. Xu, H.D., J. Feng, Z.X. Guo, Y.J. Ou, and J.Y. Wang. 2009. Detection of red-spotted grouper nervous necrosis virus by loop-mediated isothermal amplification. J. Virol. Methods 163, 123-128.
  13. Zhang, F., L. Ma, X. Xu, J. Zheng, Y. Shi, Y. Lu, and Y. Miao. 2009. Sensitive and rapid detection of Karenia mikimotoi (Dinophyceae) by loop-mediated isothermal amplification. Harmful Algae 8, 839-842. https://doi.org/10.1016/j.hal.2009.03.004