Construction of the custom-made DNA chip for pathogenic diagnosis of bacteria related with periodontal disease

用常规方法分析所有已知功能基因的表达需要花费大量时间和成本。此外，我们不能期望在未来利用任何商业化的口腔细菌微阵列。因此，我们制备了牙龈卟啉单胞菌致病性相关基因的DNA定制阵列，与成人牙周病的疾病活动密切相关，并建立了微阵列程序，如杂交，洗涤和标记条件，以及如何分析。因此，与总RNA相比，mRNA是捕捉基因表达的低信号强度的良好起始材料。此外，当我们使用定量的来自人类基因的标记外标物而不是16 S rRNA（内标物）进行芯片-芯片归一化时，微阵列和实时PCR之间的结果匹配变得很明显。对牙龈卟啉单胞菌进行环境胁迫后的转录组分析（1.生长过程中的基因表达谱; 2.氯化血红素限制下的基因表达谱; 3.氧化应激下的基因表达谱）。这些结果表明，不仅可以检测牙龈卟啉单胞菌中各种致病基因的表达，而且可以揭示致病基因之间的关系。此外，该技术预期有可能监测牙周袋中细菌的活动程度。

It takes long time and much cost to analyze the expressions of all known-functional genes by the conventional method. Furthermore, we cannot expect to utilize any commercial-based microarrays for oral bacterial in the future. Therefore, we prepared a DNA custom-made array of pathogenicity-associated genes in Porphyromonas gingivalis, strongly associated with disease activity in adult periodontal disease, and established the microarray procedure such as hybridization, wash, and labeling condition, and how to analyze. Consequently, mRNA was good start material for catching low signal intensities of gene expressions, compares with Total RNA. Moreover, it became clear to match results between microarray and Real-time PCR when we used fixed quantity of the labeled external standards from human genes for chip-chip normalization rather than 16S rRNA (internal standard). Following various environmental change was stressed to P. gingivalis and the transcriptome profiling was analyzed (1. Gene expression profiling during growth; 2. Gene expression profiling under hemin limitation; 3. Gene expression profiling under oxidization stress). These results suggested that we can expect not only to detect gene expressions of various pathogen but also to demonstrate the relationship among the pathogenic genes in P. gingivalis. Moreover, this technology expected a possibility that it could monitor of the degree of activity of the bacteria in a periodontal pocket.