目的 研究ITS序列分析和β-微管蛋白基因序列分析在曲霉菌鉴定中的临床应用价值.方法 收集2007年7月至2010年1月,首都医科大学附属北京同仁医院真菌性鼻窦炎病原菌株中曲霉菌124株,分别对其进行形态学和分子鉴定.形态学包括传统培养方法、玻片培养和乳酸酚棉蓝染色及KOH消化后显微镜镜检.将菌株的PCR扩增产物进行ITS序列分析和β-微管蛋白基因序列分析,其测序结果与GenBank、European Molecular Biology Laboratory和DNA Data Bank of Japan 3个数据库比对,得到分子鉴定结果.结果 形态学鉴定为黄曲霉的56株曲霉中,经ITS序列分析鉴定为黄曲霉55株,寄生曲霉1株,β-微管蛋白基因序列分析结果与ITS序列分析相同;形态学鉴定为烟曲霉的37株曲霉中,ITS序列分析鉴定为37株烟曲霉复合种,β-微管蛋白基因序列分析鉴定为烟曲霉35株和仑图卢斯曲霉2株;形态学鉴定为杂色曲霉的21株曲霉中,ITS序列分析鉴定为杂色曲霉16株和未鉴定到种的曲霉5株,β-微管蛋白基因序列分析鉴定为杂色曲霉16株和聚多曲霉5株;形态学鉴定为构巢曲霉的10株曲霉中,ITS序列分析和β-微管蛋白基因序列分析均鉴定为构巢曲霉.结论 β-微管蛋白基因序列分析曲霉的分辨率较ITS序列分析高,可以将曲霉准确鉴定到种,ITS序列可以分析到曲霉复合种.
Background Accurate identification of bacterial isolates is an essential task in clinical microbiology. This study compared culturing to analyzing 16S rRNA gene sequences as methods to identify bacteria in clinical samples. We developed a key technique to directly identify bacteria in clinical samples via nucleic acid sequences, thus improving the ability to confirm pathogens.Methods We obtained 225 samples from Beijing Tongran Hospital and examined them by conventional culture and 16S rDNA sequencing to identify pathogens. This study made use of a modified sample pre-treatment technique which came from our laboratory to extract DNA. 16S rDNA was amplified by PCR. The amplified product was sequenced on a CEQ8000 capillary sequencer. Sequences were uploaded to the GenBank BLAST database for comparison.Results Among the positively cultivated bacterial strains, seven strains were identified differently by Vitek32 and by 16S rDNA sequencing. Twelve samples that were negative by standard culturing were determined to have pathogens by sequence analysis.Conclusion The use of 16S rRNA gene sequencing can improve clinical microbiology by providing better identification of unidentified bacteria or providing reference identification of unusual strains.
