Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Development of Prevotella nigrescens ATCC $33563^T$-Specific PCR Primers

Prevotella nigrescens ATCC $33563^T$ 균주-특이 중합효소연쇄반응 프라이머 개발

  • Song, Soo-Keun (Department of Oral Biochemistry, Medical college, Chosun University) ;
  • Yoo, So-Young (Department of Oral Biochemistry, Medical college, Chosun University) ;
  • Kim, Mi-Kwang (Department of Oral Biochemistry, Medical college, Chosun University) ;
  • Kim, Hwa-Sook (Chunnam Techno College, Gokseong County) ;
  • Lim, Sun-A (Chunnam Techno College, Gokseong County) ;
  • Kim, Do-Kyung (Department of Oral Physiology, Medical college, Chosun University) ;
  • Park, Jae-Yoon (Department of Biochemistry & Molecular Biology, Medical college, Chosun University) ;
  • Kook, Joong-Ki (Department of Oral Biochemistry, Medical college, Chosun University)
  • 송수근 (조선대학교 치과대학 구강생화학교실) ;
  • 유소영 (조선대학교 치과대학 구강생화학교실) ;
  • 김미광 (조선대학교 치과대학 구강생화학교실) ;
  • 김화숙 (전남과학대학 치위생과) ;
  • 임선아 (전남과학대학 치위생과) ;
  • 김도경 (조선대학교 치과대학 구강생리학교실) ;
  • 박재윤 (조선대학교 의과대학 생화학.분자생물학교실) ;
  • 국중기 (조선대학교 치과대학 구강생화학교실)
  • Published : 2008.09.30
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Abstract

A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 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 Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain.

본 연구는 Prevotella nigrescens ATCC $33563^T$에 대한 균주 특이 DNA 프로브라고 보고된 Pn10 프로브의 균주 특이성을 한국인에서 분리된 P. nigrescens의 임상분리 균주를 이용하여 검증하고, P. nigrescens ATCC $33563^T$ 균주 특이 PCR 프라이머를 개발하고자 시행되었다. P. nigrescens와 유전학적으로 가장 가까운 Prevotella intermedia를 포함한 구강 내 치주질환 원인균종인 5균종의 표준균주 및 참고균주, 그리고 P. nigrescens와 P. intermedia의 임상분리 균주를 이용하여 Southern blot 분석법을 시행하였다. Southern blot 분석 결과 Pn10 DNA 프로브에 P. nigrescens ATCC $33563^T$ 및 ChDC KB6 두 균주 지놈 DNA가 검출되었다. P. nigrescens KB6 균주에서 Pn10 DNA 프로브와 상동성이 있는 부위를 PCR법으로 증폭(KB6-Pn10)하여 클로닝한 다음 Pn10 DNA프로브와 같이 핵산 염기서열을 결정하여 상동성을 비교하였다. 그 결과 Pn10과 KB6-Pn10의 핵산염기서열간의 Percent identity는 98.8%였으며, divergence는 0.6%였다. Pn10 DNA 프로브의 핵산염기서열을 바탕으로 두 중류 프라이머 쌍(Pn10-F-AC/Pn10-R-AC 및 Pn10-F-A/Pn10-R-A)을 설계 및 제작하여 P. nigrescens ATCC $33563^T$에 대한 균주 특이성을 PCR법으로 검증하였다. 이들 프라이머 쌍들의 민감도(sensitivity) 조사 결과, 이들은 P. nigrescens ATCC $33563^T$ 지놈 DNA 4 pg까지 검출할 수 있음을 알았다. 이상의 연구 결과를 종합하면, Pn10 DNA 핵산염기서열을 바탕으로 설계된 Pn10-F-AC/Pn10-R-AC 및 Pn10-F-A/Pn10-R-A 프라이머 쌍들은 P. nigrescens ATCC $33563^T$를 신속 정확하게 검출하는 수 있어, 균주의 보존적 측면에서 유용하게 이용될 수 있을 것으로 생각된다.

Keywords

References

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