Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide.
Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide.
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DOI：
10.1186/1471-2180-13-157
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发表时间：
2013-07-13
期刊：
影响因子：
4.2
通讯作者：
Ansai T
中科院分区：
文献类型：
--
作者：
Yasunaga A;Yoshida A;Morikawa K;Maki K;Nakamura S;Soh I;Awano S;Ansai T
变形链球菌和红链球菌与人类龋齿的发生有关。然而，以前的诊断系统不适合监测口腔标本中的活细胞数量。评估存活和死亡细菌细胞数量与口腔状况之间的关系对于了解口腔传染病具有重要意义。据报道，单叠氮丙啶（PMA）可以穿透死细胞后的膜损伤和交联DNA，从而抑制DNA扩增。在本研究中，我们建立了PMA结合实时荧光定量PCR （PMA- qpcr）对两种活的人类致龋病原体S. mutans和S. sobrinus进行选择性分析的方法。我们设计了变形链球菌和sobrinus的物种特异性引物，生成了测量细胞数量的标准曲线，并评估了该试验的动态范围。为了确定该试验的有效性，将PMA添加到活细胞和高压灭菌的细胞混合物中。PMA处理有效地阻止了死细胞的DNA扩增。在这些生物体中没有观察到来自死细胞的DNA扩增。此外，我们应用该方法分析口腔标本中的活细胞数。在无龋患者中，唾液中变形链球菌存活细胞的数量与牙菌斑中变形链球菌存活细胞的数量有显著的相关性，而唾液与龋齿本质之间没有相关性。龋阳性唾液的总细胞数和活细胞数明显高于无龋唾液。最后，我们分析了该方法在体外口腔生物膜分析中的实用性。我们采用PMA-qPCR技术检测了过氧化氢处理的浮游细胞和口腔生物膜中活菌的数量。在浮游细胞中，随着H2O2浓度的增加，活细胞数量显著减少，而生物膜细胞数量仅略有下降。PMA-qPCR可用于定量口腔标本中活的致龋病原体，并适用于口腔生物膜实验。该试验将有助于阐明口腔标本中活细胞数量与口腔状况之间的关系。
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). 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. 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.
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影响因子：
--
作者：
LINDQUIST, B;EMILSON, CG;WENNERHOLM, K
通讯作者：
WENNERHOLM, K
影响因子：
9.4
作者：
Yoshida, A;Suzuki, N;Koga, T
通讯作者：
Koga, T
DOI：
10.1111/j.1600-0528.1991.tb00131.x
发表时间：
1991-06-01
影响因子：
2.3
作者：
FUJIWARA, T;SASADA, E;OOSHIMA, T
通讯作者：
OOSHIMA, T
影响因子：
3
作者：
Okada, M;Soda, Y;Kozai, K
通讯作者：
Kozai, K
影响因子：
4.4
作者：
Pan, Y.;Breidt, F., Jr.
通讯作者：
Breidt, F., Jr.