García L, Tercero J C, Legido B, Ramos J A, Alemany J, Sanz M
Departamento de Bioquímica, Facultad de Medicina, Universidad Complutense de Madrid, Spain.
J Periodontal Res. 1998 Jan;33(1):59-64. doi: 10.1111/j.1600-0765.1998.tb02292.x.
The identification of specific periodontal pathogens by conventional methods, mainly anaerobic cultivation, is difficult, time consuming and even sometimes unreliable. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.), Porphyromona gingivalis (P.g.) and Prevotella intermedia (P.i.) was developed for rapid and easy identification of these specific bacterial pathogens in subgingival plaque samples. In this paper, there is a detailed description of the oligonucleotide primer selection, DNA extraction and PCR conditions and the sequencing of the amplified products. The locus chosen to be amplified is a highly variable region in the 16S ribosomal DNA. For the development of this technique ATCC cultures and pure cultures from subgingival plaque samples taken from periodontitis patients were used. As an internal positive control a recombinant plasmid was developed. This simple DNA extraction procedure and the DNA amplification and visualization of the amplified product permits the detection of the bacteria in a working day. Thus, this multiplex PCR method is a rapid and effective detection method for specific periodontal pathogens.
通过传统方法（主要是厌氧培养）鉴定特定的牙周病原体既困难、耗时，甚至有时还不可靠。因此，开发了一种多重PCR方法，用于同时检测伴放线放线杆菌（A.a.）、牙龈卟啉单胞菌（P.g.）和中间普氏菌（P.i.），以便快速、简便地鉴定龈下菌斑样本中的这些特定细菌病原体。本文详细描述了寡核苷酸引物的选择、DNA提取、PCR条件以及扩增产物的测序。选择扩增的基因座是16S核糖体DNA中的一个高度可变区域。为了开发这项技术，使用了来自牙周炎患者龈下菌斑样本的ATCC培养物和纯培养物。作为内部阳性对照，构建了一个重组质粒。这种简单的DNA提取程序以及DNA扩增和扩增产物的可视化使得能够在一个工作日内检测到细菌。因此，这种多重PCR方法是一种快速、有效的特定牙周病原体检测方法。
