优化定量聚合酶链反应以定量复杂菌斑生物膜中的牙周病原体
Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm.
作者信息
Kirakodu S S, Govindaswami M, Novak M J, Ebersole J L, Novak K F
机构信息
Center for Oral Health Research, College of Dentistry, University of Kentucky College of Dentistry, Lexington, USA.
出版信息
Open Dent J. 2008;2:49-55. doi: 10.2174/1874210600802010049. Epub 2008 Mar 11.
Abstract
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.
摘要
定量聚合酶链反应（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，目前所使用的qPCR方案存在差异。本研究旨在评估其中两个变量的有效性，以期开发出一种更标准化的qPCR方案。所评估的两个变量为：（1）在混合菌斑样本中，使用DNA含量与实际细胞计数来估计细菌数量；（2）三种不同的通用引物与种特异性引物在扩增这些样本中特定目标病原体方面的有效性。结果促成了一种标准化方案的开发，该方案显示出高度的可重复性，变异系数较低即证明了这一点。结果还证实，这种标准化的qPCR方案可作为一种灵敏的方法，用于量化人类菌斑样本中的特定细菌种类。
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