Development of discriminative and quantitative detection method for live and dead oral bacteria

Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important forunderstanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR). We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves 機関番号：27102研究種目： 基盤研究(C)研究期間： 平成22年度～平成24年度 課題番号：22592341研究課題名（和文） 生菌・死菌の区別が可能な新しい定量的口腔細菌検出法の開発研究課題名（英文） Development of discriminative and quantitative detection method for live and dead o … More ral bacteria研究代表者 吉田明弘（YOSHIDA, AKIHIRO）九州歯科大学・歯学部・助教研究者番号：20364151 for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. In addition, we applied this assay to analyze viable cell numbers in oral specimens. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers. PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens andthe oral status. Less

Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important forunderstanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR). We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves 機関番号：27102研究種目： 基盤研究(C)研究期間： 平成22年度～平成24年度 課題番号：22592341研究課題名（和文） 生菌・死菌の区別が可能な新しい定量的口腔細菌検出法の開発研究課題名（英文） Development of discriminative and quantitative detection method for live and dead o … More ral bacteria研究代表者 吉田明弘（YOSHIDA, AKIHIRO）九州歯科大学・歯学部・助教研究者番号：20364151 for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. In addition, we applied this assay to analyze viable cell numbers in oral specimens. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers. PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens andthe oral status. Less