应用16S rDNA结合膜芯片检测3种产黑色素牙周可疑致病菌

目的:探讨采用16SrDNA结合膜芯片检测牙龈卟啉菌Pg、中间普氏菌Pi和变黑普氏菌Pn的价值。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、Pi和Pn的16SrDNA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,PCR产物和已点样有Pg、Pi和Pn的3种探针的膜芯片杂交,进行分析。结果:膜芯片上的Pg、Pi和Pn3种探针只能与相对应的Pg、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:用16SrDNA结合膜芯片,可准确检测Pg、Pi和Pn,具有很高的特异性,有望成为一种有效的临床检测方法。

Identification of P. gingivalis P. intermedia P. nigrescens by 16S rDNA and membrane microarray chip

PURPOSE: The aim of this study is to identify three kinds of black-pigmented periodontal pathogens P. gingivalis Pg, P. intermedia Pi, P. nigrescens Pn by 16S rDNA and microarray. METHODS: A pair of universal primers which can amplify a section of conservative domain of bacterial 16S rDNA based on the sequences of 16S rDNA in Genebank were designed. Then the specific oligonucleotide probes for Pg Pi Pn based on the sequences of the conservative domain were constructed. Standard bacterial genomic DNAs were amplified using the designed universal primers by PCR , and labeled by digoxigenin at the same time, the products of PCR were hybridized with the microarray in which the specific probes were added. The results of hybridization were analysed. RESULTS: The results of hybridization showed that the specific probes of Pg Pi Pn on microarray reacted only with corresponding PCR products of Pg Pi Pn, not reacted with others. CONCLUSION: The method of 16S rDNA and membrane microarray could be useful to identify Pg Pi Pn, and had high specificity. It will be developed into a kind of clinical bacterial detective system. Supported by National Natural Science Foundation of China (Grant No. 30271413) and National Tenth Five-year Science and Technology Key Project(Grant No.2004BA720A24).