细菌核糖体23S亚单位基因芯片在感染性疾病快速诊断中的应用研究

23S Ribosomal DNA Microchips Applied to Detect Clinical Pathogens in Rapid Diagnosis of Infectious Diseases

目的研究建立快速检测细菌DNA的方法,建立含有10种细菌探针的检测用基因芯片模型。方法使用合成的扩增细菌核糖体23 S亚单位(23S rDNA)寡核苷酸探针,制备基因芯片;设计23S rDNA通用引物,应用PCR(聚合酶链反应)法,扩增细菌核糖体23S亚单位基因(23S rDNA),扩增后产物与芯片上的探针杂交,用荧光扫描仪检测信号;对23种临床常见致病菌、培养物及临床病例标本采用本方法进行检测,并与常规细菌培养法比较。结果基因芯片检测临床致病菌具有较高的特异性和灵敏性。结论利用寡核苷酸探针基因芯片检测系统具有一定的种属鉴别能力,比常规培养法快速、准确。

OBJECTIVE To investigate a rapid method for detecting clinical bacterial 23S ribosomal DNA （23S rDNA） and to develop a gene chip model containing 10 probes for pathogens. METHODS Ten synthesized oligonucleotide 23S rDNA probes were employed to make gene chips by spoter. The DNA of bacteria was amplified by 23S rDNA universal primers and the PCR product was then applied to the gene chips for hybridization. A fluorescent scanner was used to observe and record the hybridization signals. Compared with routine bacterial culture method, this novel PCR method was used to detect 23S rDNA of 23 kinds of pure bacteria, culture bottles and clinical specimens. RESULTS It showed better sensitivity and specificity in detecting bacterial 23S rDNA by gene chips method. CONCLUSIONS The oligonucleotide microarray could determine most of the tested strains at species level. It is more rapid and accurate than routine culture method. The overall time for sample process, hybridization and data acquisition lasts about 4-5 hours.