Pn10 DNA 프로브를 바탕으로 한 Prevotella nigrescens ATCC 33563^(T) 균주-특이 중합효소연쇄반응 프라이머 개발

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 the Pn10 DNA probe as 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 1875 bp and 1873 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.