Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Nested PCR for the Detection of Streptococcus mutans

Nested PCR를 이용한 Streptococcus mutans의 검출

  • Choi, Min-Ho (Department of Prosthodontics, College of Dentistry, Chosun University) ;
  • Yoo, So-Young (Department of Oral Biochemistry, College of Dentistry, Chosun University) ;
  • Lim, Chae-Kwang (Institute of Forensic Odontology, College of Dentistry, Chosun University) ;
  • Kang, Dong-Wan (Department of Prosthodontics, College of Dentistry, Chosun University) ;
  • Kook, Joong-Ki (Department of Oral Biochemistry, College of Dentistry, Chosun University)
  • 최민호 (조선대학교 치과대학 보철학교실) ;
  • 유소영 (조선대학교 치과대학 구강생화학교실) ;
  • 임채광 (조선대학교 치과대학 법의치학연구소) ;
  • 강동완 (조선대학교 치과대학 보철학교실) ;
  • 국중기 (조선대학교 치과대학 구강생화학교실)
  • Published : 2006.03.01
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Abstract

This study was undertaken to develop PCR primers for the identification and detection of Streptococcus mutans (by)using species-specific forward and universal reverse primers. These primers targeted the variable regions of the 16S ribosomal RNA coding gene (rDNA). The primer specificity was tested against 11S. mutans strains and 10 different species (22 strains) of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of S. mutans ATCC $25175^T$. The data showed that species-specific amplicons were obtained from all the S. mutans strains tested, which was not observed in the other species. The direct and nested PCR could detect as little as 2 pg and 2 fg of the chromosomal DNA from S. mutans ATCC $25175^T$, respectively. This shows that the PCR primers are highly sensitive and applicable to the detection and identification of S. mutans.

치아우식중의 원인균 중 하나인 Streptococcus mutans를 종 수준에서 동정할 수 있는 중합효소연쇄반응 프라이머를 개발하기 위하여 본 연구를 시행하였다. 표준균주인 S. mutans ATCC $25175^T$및 본 연구에서 이용된 S. mutans 균주들의 16S rRNA 유전자 핵산염기서열을 바탕으로 S. mutans 종-특이 중합효소연쇄반응 프라이머(ChDC-SmF2와 ChDC-SmR2)를 설계 및 제작하였다. 프라이머의 특이도는 S. mutans 11균주와 구강 내 존재하는 12세 균 종(22균주)을 대상으로 실시하였다. 프라이머의 민감도는표준균주인 S. mutans ATCC $25175^T$의 유전체 DNA를 추출하여 실시하였다. 프라이머의 특이도 실험결과 10균주의 S. mutans 유전체 DNA에서만 종-특이 중합효소 연쇄반응 산물이 증폭되었고, 다른세균종의 유전체 DNA에서는 증폭되지 않았다. 또한 감수성 실험 결과 본 연구에서 사용된 프라이머는 direct PCR에 의해 S. mutans ATCC $25175T\^T$ 유전체 DNA 100 pg까지 검출 할 수 있었다. 또한 27F와 1492R 프라이머로 16S rDNA를 먼저 증폭한 다음, 이 증폭물 10배 회식한 것을 표적으로 해서 nested PCR을 시행한 결과 S. mutans ATCC $25175^T$ 유전체 DNA를 2 fg까지 검출할 수 있었다. 이상의 결과를 종합할 때, ChDC-SmF2와 ChDC-SmR2 프라이머들은 S. mutans 균주 유전체 DNA에 대한 높은 민감도를 가지고 있으며, 종-특이적으로 동정 및 검출하는 데 이용될 수 있을 것으로 생각된다.

Keywords

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