Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Development of prevotella intermedia ATCC 49046 Strain-Specific PCR Primer Based on a Pig6 DNA Probe

Pig6 DNA probe를 기반으로 하는 Prevotella intermedia ATCC 49046 균주-특이 PCR primer 개발

  • Jeong Seung-U (Department of Periodontology, College of Dentistry, Chosun University) ;
  • Yoo So-Young (Department of Oral Biochemistry, College of Dentistry, Chosun University) ;
  • Kang Sook-Jin (Department of Oral Biochemistry, College of Dentistry, Chosun University) ;
  • Kim Mi-Kwang (Department of Oral Biochemistry, College of Dentistry, Chosun University) ;
  • Jang Hyun-Seon (Department of Periodontology, College of Dentistry, Chosun University) ;
  • Lee Kwang-Yong (Undergraduate student, College of Dentistry, Chosun University) ;
  • Kim Byung-Ok (Department of Periodontology, College of Dentistry, Chosun University) ;
  • Kook Joong-Ki (Department of Oral Biochemistry, College of Dentistry, Chosun University)
  • 정승우 (조선대학교 치과대학 치주과학교실) ;
  • 유소영 (조선대학교 치과대학 구강생화학교실) ;
  • 강숙진 (조선대학교 치과대학 구강생화학교실) ;
  • 김미광 (조선대학교 치과대학 구강생화학교실) ;
  • 장현선 (조선대학교 치과대학 치주과학교실) ;
  • 이광용 (조선대학교 치과대학 치의학과) ;
  • 김병옥 (조선대학교 치과대학 치주과학교실) ;
  • 국중기 (조선대학교 치과대학 구강생화학교실)
  • Published : 2006.06.01
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Abstract

The purpose of this study is to develop the strain-specific PCR primers for the identification of prevotella inter-media ATCC 49046 which is frequently used in the pathogenesis studies of periodontitis. The Hind III-digested genomic DNA of P. intermedia ATCC 49046 were cloned by random cloning method. The specificity of cloned DNA fragments were determined by Southern blot analysis. The nucleotide sequence of cloned DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pig6 DNA probe were hybridized with the genomic DNA from P. intermedia strains (ATCC $25611^T$ and 49046) isolated from the Westerns, not the strains isolated from Koreans. The Pig6 DNA probe were consisted of 813 bp. Pig6-F3 and Pig6-R3 primers, designed base on the nucleotide Sequences Of Pig6 DNA Probe, were 3150 specific to the only both P. intermedia ATCC $25611^T$ and P. intermedia ATCC 49046. In the other hand, Pig6-60F and Pig6-770R primers were specific to the only P. intermedia ATCC 49046. The two PCR primer sets could detect as little as 4 pg of chromosomal DNA of P. intermedia. These results indicate that Pig6-60F and Pig6-770R primers have proven useful for the identification of P. intermedia ATCC 49046, especially with regard to the maintenance of the strain.

본 연구는 치주질환 병인론 연구에 빈번히 사용되는 Prevotella intermedia ATCC 49046 균주를 특이적으로 검출 및 동정할 수 있는 PCR primer를 개발하기 위하여 시행하였다. P. intermedia ATCC 49046 유전체 DNA를 추출하고, Hind III 제한효소를 이용하여 무작위 클로닝법으로 유전체 DNA 절편을 얻었다. Southern blot 분석법을 이용하여 DNA 절편의 특이성을 조사하였고, chain termination 법을 이용하여 핵산염기서열을 결정하였다. 이를 바탕으로 PCR primer를 설계하고, P. intermedia ATCC 49046에 대한 균주 특이성 및 검출 한계(민감도)를 조사하였다. Southern blot 분석 결과 Pig6 DNA probe는 서양인에서 분리 동정된 P. intermedia 균주와만 hybridization하였고, 한국인에서 분리 동정된 P. intermedia균주들과는 반응이 없었다. Pig6 DNA probe는 813 bp의 핵산염기로 구성되어 있었으며, 이를 바탕으로 설계된 Pig6-F3와 Pig6-R3 primer 쌍에 의해서는 서양 균주에 특이적인 PCR산물이 증폭되었다. Pig6-60F와 Pig6-770R primer 쌍에 의해서는 P. intermedia ATCC 49046 유전체 DNA에서만 특이적인 PCR 산물이 증폭되었다. 두 가지 primer 쌍들 각각에 대한 P. intermedia 유전체 DNA량의 검출 한계를 알아보기 위한 민감도실험 결과 두가지 primer 쌍들 모두 4 pg (약2000마리)까지 검출 가능하였다. 이상의 연구결과를 종합하면, Pig6-60F와 Pig6-770R primer쌍은 P. intermedia ATCC 49046을 균주 특이적으로 동정할 수 있어, 이 균주의 보존적 측면에서 유용하게 이용될 수 있을 것으로 사료된다.

Keywords

References

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