16SrDNA寡核苷酸芯片鉴定致病菌的初步研究

Development of 16S rDNA oligonucleotide microarray for identification of pathogenic bacteria

目的:建立一种基于 16SrDNA寡核苷酸芯片的检测和鉴定常见致病菌的技术。方法:以 16SrDNA为靶标,针对待检细菌设计合成一系列寡核苷酸探针,制备寡核苷酸芯片。细菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱进行分析归纳,得到一套种和属特异的检测模式。结果:以本室保存的 33株菌 (包括 7个属 15个种)进行初步检验,结果表明,种水平上的鉴定准确率为 78. 79%, 21. 21%可鉴定到属的水平。结论:该研究建立的 16SrDNA寡核苷酸芯片技术可以稳定、特异地实现细菌的高通量鉴定,为进一步检测研究奠定了基础。

Objective: To develop an oligonucleotide microarray containing 26 species-specific probes for identification of medically important pathogenic bacteria. Methods: Oligonucleotide probes were designed and synthesized to create an oligonucleotide microarray by Flexys high density arrayer. The DNA of bacteria was amplified by universal primers targeted to 16S rDNA and the PCR product was hybridized with the oligonucleotide microarray. Axon 4100A scanner was used to detect the fluorescent signals. Sixty strains of bacteria in pure culture belonged to 15 species were applied to the oligonucleotide microarray and a series of specific hybridization profiles corresponding to each species were obtained. Results: Thirty-three strains were used to test the reliability of the microarray system, of which 78.79% were correctly identified to the species level, and 21.21% to the genus level.Conclusion: The oligonucleotide microarray system was proved to be a stable and efficient universal identification system for pathogenic bacteria and can be further extended to a wider range of pathogenic bacteria by adding more oligonucleotide probes.