Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm.
Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm.
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DOI：
10.2174/1874210600802010049
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Novak KF
中科院分区：
文献类型：
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作者：
Kirakodu SS;Govindaswami M;Novak MJ;Ebersole JL;Novak KF
定量PCR（qPCR）最近已被用于量化复杂群落中的微生物，包括牙菌斑生物膜。然而，所使用的qPCR方案存在变异性。本研究旨在评价其中两个变量的有效性，旨在开发更标准化的qPCR方案。评价的两个变量是（1）使用DNA含量与实际细胞计数来估计混合菌斑样本中的细菌数量，以及（2）三种不同的通用引物与种属特异性引物在扩增这些样本中的特异性靶病原体中的有效性。结果导致标准化方案的开发，该方案被证明是高度可重复的，如低变异系数所证明的。结果还证实，该标准化qPCR方案可用作定量人体菌斑样本中特定细菌物种的灵敏方法。
Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples.