【作者】 唐子圣； 曹慧敏； 刘正； 赵淑娟； 王蓓； 赵建龙； 梁景平；

【Author】 TANG Zi-sheng, CAO Hui-min, LIU Zheng, ZHAO Shu-juan, WANG Bei, ZHAO Jian-long, LIANG Jing-ping.(Department of oral Medicine, College of Stomatology, Ninth People’ s Hospital, School of Medicine, Shanghai Jiao Tong University. Shanghai 200011; Lab of Biochip, Shanghai Institute of Microsystem and Information Technology,Chinese Academy of Sciences. Shanghai 200050, China)

【机构】 上海交通大学医学院附属第九人民医院口腔内科； 中国科学院上海微系统与信息技术研究所生物芯片实验室；

【摘要】 目的:牙髓卟啉菌Pe、牙龈卟啉菌Pg、中间普氏菌Pi和变黑普氏菌Pn是4种公认的根管致病菌,因为它们培养时都能产生黑色素并且在分类学上关系密切,因此在临床上要同时检测并鉴定出这4种产黑色素根管致病菌相当困难,本研究目的就是想利用微阵列芯片技术研制一种能同时检测上述4种产黑包素根管致病菌的微阵列芯片。方法:利用Genebank中细菌16S rDNA保守区序列设计一对通用引物,通过已知Pe、Pg、Pi和Pn的16S rDNA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Pe、Pg、Pi和Pn四种探针的微阵列芯片杂交,通过对杂交结果的分析,可以不断调整探针序列直到其特异性得到证实。然后通过检测临床感染根管细菌学标本来对这个微阵列芯片进行测试。结果:杂交结果显示芯片上的Pe、Pg、Pi和Pn4种探针只能和相对应的Pe、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,并且用此芯片能从临床感染根管细菌学标本中准确快速的检测出Pe、Pg、Pi和Pn。结论:用微阵列芯片的方法可以准确检测Pe、Pg、Pi和Pn,具有很高的特异性,有希望发展为一种有效的临床检测系统。更多还原

【Abstract】 OBJECTIVE: Black-pigmented bacteria have been associated with endodontic infections. P. endodontatis Pe, P. gingivalis Pg, P. intermedia Pi and P. nigrescens Pn are four putative endodontopathogens. Since they are all black-pigmented and close in taxology. these four infected root canal pathogens are difficult identification in clinical together. The aim of this study was to develop a microarray to detect these four black-pigmented bacteria in clinical. METHODS: We designed a pair of universal primers which can amplify a section of conservative domain of bacterial 16S rDNA based the sequences of 16S rDNA in Genebank. Then we designed the specific oligonucleotide probes for Pe Pg Pi Pn based the sequences of the conservative domain. Standard bacterial genomic DNAs were amplified using the designed universal primers by PCR , and labeled by cy5 at the same time, the products of PCR hybridized with the microarray in which the specific probes were added. The results of hybridization were screened by confocal laser scanner. From the results we could adjust the sequences of these probes until the specificity of probe was comfirmed. Then this microarray was tested by detecting samples of infected root canal in clinical. RESULTS: The results of hybridization showed that the specific probes of Pe Pg Pi Pn on microarray reacted only with corresponding PCR products of Pe Pg Pi Pn, not react with others. Pe Pg Pi Pn could be identificated accurately and rapidly from root canal microbial samples by this microarray. CONCLUSION: The method of 16S rDNA and microarray could be useful to identifify Pe,Pg,Pi,Pn and clinically, and had high specificity. It will be developed into a kind of clinical bacterial detective system through adding probe quantity of oral bacteria in the panel.更多还原