Korean Journal of Microbiology (미생물학회지)

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

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

  • 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)
  • 김정구 (국립농업과학원 유전자분석개발과) ;
  • 노지나 (경기대학교 일반대학원 생명과학과) ;
  • 박동석 (국립농업과학원 유전자분석개발과) ;
  • 윤병수 (경기대학교 일반대학원 생명과학과)
  • Received : 2011.04.28
  • Accepted : 2011.06.15
  • Published : 2011.06.30
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Abstract

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.

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.

Keywords

References

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