論文 - 詳細
| RRC ID | 29392 |
|---|---|
| 著者 | Yasunaga A, Yoshida A, Morikawa K, Maki K, Nakamura S, Soh I, Awano S, Ansai T. |
| タイトル | Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide. |
| ジャーナル | BMC Microbiol |
| Abstract |
BACKGROUND: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 for understanding 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). RESULTS:We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves 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. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. 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. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. 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. CONCLUSIONS: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 and the oral status. |
| 巻・号 | 13 |
| ページ | 157 |
| 公開日 | 2013-7-13 |
| DOI | 10.1186/1471-2180-13-157 |
| PII | 1471-2180-13-157 |
| PMID | 23848601 |
| PMC | PMC3717283 |
| MeSH | Azides / metabolism Bacterial Load / methods Biofilms* Child Child, Preschool DNA Primers / genetics Female Humans Male Microbial Viability* Mouth / microbiology* Propidium / analogs & derivatives Propidium / metabolism Real-Time Polymerase Chain Reaction / methods* Staining and Labeling / methods* Streptococcus mutans / isolation & purification Streptococcus mutans / physiology* Streptococcus sobrinus / isolation & purification Streptococcus sobrinus / physiology* |
| IF | 2.989 |
| 引用数 | 22 |
| WOS 分野 | MICROBIOLOGY |
| リソース情報 | |
| 一般微生物 | JCM 1136 JCM 1140 JCM 1561 JCM 1563 JCM 6290 JCM 6331 JCM 8132 JCM 8134 JCM 8135 JCM 8350 JCM 8525 JCM 8530 JCM 8577 |