基因芯片技术检测鉴定临床常见致病真菌的初步研究

目的为了快速、简便、高通量地鉴定临床常见致病真菌,建立了一种采用基因芯片技术对临床常见的致病真菌鉴定的分子生物学方法。方法以5.8S rDNA与28S rDNA间的内转录间区2(internal transcribed spacer-2,ITS-2)为靶标,针对待检的临床常见致病真菌设计合成一系列寡核苷酸探针,制成寡核苷酸芯片。待检真菌DNA经通用引物扩增标记后,与芯片杂交,对杂交图谱分析归纳,得到一套种特异性的典型杂交图谱。待检的样品菌与基因芯片杂交,得到的杂交结果与典型图谱比对即可判断出样品的种类。结果以涉及8个属20个种的标准致病真菌菌株对芯片的特异性、重复性、灵敏度进行考察,结果表明,该研究建立的基因芯片技术可以有效地区分20种临床常见致病真菌,特异性良好,重复性良好(信噪比CV<10%),灵敏度为15 pg/ml真菌DNA。收集从临床分离的84株致病真菌菌株对基因芯片进行试用,结果显示基因芯片的鉴定结果与常规鉴定方法的鉴定结果一致。结论这项技术的建立可以稳定、特异性地实现临床常见致病真菌的高通量鉴定,为进一步检测研究奠定了基础。

Detection and identification of fungal pathogens by oligonucleotide microarray hybridization

Objective To establish an oligonucleotide microarray system for detection and identification of fungal pathogens. Methods Oligonucleotide probes targeting to the internal transcribed spacer-2 of rDNA were designed and synthesized to create an oligonucleotide microarray. The DNA of fungi was amplified by universal primers and the PCR product was hybridized with the oligonucleotide microarray. Genepix4000B scanner was used to detect the fluorescent signals. A series of specific hybridization profiles corresponding to species were obtained. Results The fungal pathogens belonging to 20 species and 8 genera were used to test the specificity, reproducibility and sensitivity of the microarray system. The results showed that the microarray system could successfully discriminate the fungal pathogens to the species level, the specificity and stability were good, and the sensitivity was 15 pg/ml of DNA. The microarrays were used to identify 84 strains of clinical isolated and the results of identification were consistent with that by the traditional diagnostic methods such as microscopy and culture. Conclusion The oligonucleotide microarray system was proved to be a stable and efficient universal identification system for pathogenic fungi and can be further extend to a wider range of pathogenic fungi by adding more oligonucleotide probes.