【摘要】 目的:研制快速检测牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)、牙髓卟啉单胞菌(Porphyromonas endodontalis,Pe)、中间普氏菌(Prevotella intermedia,Pi)和变黑普氏菌(Prevotella nigrescens,Pn)的芯片系统。方法:利用Genebank中细菌16SrDNA保守区序列,设计一对通用引物;通过已知Pg、Pe、Pi和Pn的16SrDNA可变区序列,设计合成对应的特异性寡核苷酸探针。先用通用引物PCR扩增所有标准菌株的DNA并作荧光标记,PCR产物和已点样有Pg、Pe、Pi和Pn4种探针的芯片杂交,并用荧光扫描仪进行分析。结果:芯片上的Pg、Pe、Pi和Pn4种探针只与相对应的Pg、Pe、Pi和Pn的PCR产物反应,而与其他标准菌株的PCR产物无反应。结论:应用16SrDNA基因芯片可以准确检测Pg、Pe、Pi和Pn,快速、灵敏、特异性高,有望在临床上得到应用。更多还原

【Abstract】 Objective:To develop a rapid detection system for four kinds of black-pigmented root canal pathogens Porphyromonas gingivalis(Pg), Porphyromonas endodontalis(Pe), Prevotella intermedia (Pi), Prevotella nigrescens(Pn) by 16 S rDNA microarray. Method:A pair of universal primers which can amplify a section of conservative region of bacterial 16 S rDNA based on the sequences of 16 S rDNA in Genebank was designed. Then the specific oligonucleotide probes for Pg、Pe、Pi、Pn based on the sequences of the variable region were constructed. DNA of standard bacterial strains was amplified using the universal primers and labeled by CY5.The products of PCR were hybridized with the microarray in which the 4 specific probes were added. The results of hybridization were analyzed by the laser scanner.Result:The results of hybridization showed that the specific probes of Pg、Pe、Pi、Pn on the microarray reacted only with corresponding PCR products of Pg、Pe、Pi、Pn,not reacting with others.Conclusion:16 S rDNA microarray could be a useful method to identify Pg、Pe、Pi、Pn with high speed, sensitivity, specificity .It may be developed into a clinical detection system.更多还原