Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC 25611T. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

The aim of this study was to develop Prevotella intermedia ATCC 49046-specific PCR primers designed based on the nucleotide sequence of a DNA probe Pig28. The strainspecificity of the PCR primers, Pig28-F1/Pig28-R1, was confirmed with 9 strains of P. intermedia and 25 strains (15 species) of Prevotella species. The detection limit of the PCR primers was 2 pg of the purified genomic DNA of P. intermedia ATCC 49046. These PCR primers were found to be useful for identifying P. intermedia ATCC 49046, particularly for determining the authenticity of the strain.

The DNA probes Pn17 and Pn34 were evaluated for their ability to specifically detect clinical strains of P. intermedia and P. nigrescens from a Korean population by dot blot hybridization. These probes were sequenced by extension termination and their specificity was determined by Southern blot analysis. The results revealed that the Pn17 sequence (2,517 bp) partially encodes an RNA polymerase beta subunit (rpoB) and that Pn34 (1,918 bp) partially encodes both rpoB (1-169 nts) and the RNA polymerase beta subunit (rpoB'; 695-1918 nts). These probes hybridized with both HindIII- and PstI-digested genomic DNAs from the strains of P. intermedia and P. nigrescens used in this study. Interestingly, each of the hybrid bands generated from the HindIII-digested genomic DNAs of the two bacterial species could be used to distinguish between them via restriction fragment length polymorphism. These results thus indicate that Pn17 and Pn34 can simultaneously detect P. intermedia and P. nigrescens.

치아의 세균들은 잇몸에서 염증반응을 일으켜서 치은염과 치주염같은 잇몸 질환의 원인이 된다. 따라서 잇몸질환의 예방과 치료를 위해서는 치아 세균에 의한 염증반응을 조절하는 것이 가장 효과적인 방법이다. 현재 대부분의 구강 관리 제품들은 살균제를 이용하여 구강 세균을 제거하는 방법을 사용하고 있다. 하지만 최근의 연구들은 심지어 열 처리로 사멸된 구강 세균도 염증반응을 일으킬 수 있다는 사실을 보고하고 있다. 따라서 보다 효과적인 잇몸 염증반응 억제를 위해서는 살균제를 이용한 방법에 대한 개선이 필요하다. 또한 아직까지 구강 세균에 의한 잇몸 염증반응의 기작과 효과적인 천연 염증 억제 물질들은 보고되어 있지 않다. 본 연구에서는 대표적인 치은염, 치주염 유발세균인 Prevotella intermedia와 인간의 잇몸상피세포를 이용하여, 실제로 잇몸에서 일어나는 염증반응의 기작을 연구하고, 이를 통해 효과적인 천연 잇몸 염증 완화 물질을 도출하려고 하였다. 실험 결과, Prevotella intermedia는 잇몸상피세포를 자극하여 염증매개인자인 IL-8을 분비하게 함으로써 잇몸 염증반응을 개시하였다. 또한 Prevotella intermedia에 의한잇몸 염증반응은 기전적으로 COX-2, AP-1, TNF-α와 연관되어 있었으며, 녹차추출물은 Prevotella intermedia에 의한 잇몸 염증반응을 효과적으로 억제할 수 있음을 확인하였다. 따라서 본 연구는 치아 세균에 의한 잇몸 염증반응의 기전 연구를 통해서 효과적인 천연 잇몸질환 개선 물질을 도출했다는 점에서 중요한 의미를 가진다.