8种深部真菌DNA微阵列鉴定方法的建立及应用  

Development of a Oligonucleotide Microarray for Detection of Eight Common Deep Fungi

作　　者：胡一敏[1] 周新[1] 郑璇[1] 谢焱[1] 刘松梅[1] 鲁双燕[1] 黎四维[1] 

机构地区：[1]武汉大学中南医院基因诊断中心,武汉430071

出　　处：《现代检验医学杂志》2011年第3期30-34,共5页Journal of Modern Laboratory Medicine

基　　金：基金项目：国家自然基金资助项目,编号30672012.

摘　　要：目的建立一种能快速鏊定临床常见8种深部真菌的DNA微阵列。方法选取临床常见的8种真菌，包括白色假丝酵母、光滑假丝酵母、热带假丝酵母、近平滑假丝酵母、土曲霉、黄曲霉、烟曲霉和米根霉。利用通用引物扩增真菌ITS区，生物素标记目的基因。针对扩增靶序列的可变区段设计探针，以尼龙膜为载体，建立DNA微阵列。采用所建立的DNA微阵列对45株临床真菌分离株进行检测。结果应用DNA微阵列技术成功地对8种临床常见真菌菌种进行了检测，检测时间可缩短至8h。白色假丝酵母、光滑假丝酵母、热带假丝酵母、近平滑假丝酵母、米根霉、黄曲霉以及土由霉的探针的检测特异度高，无非特异性反应；烟曲霉的检测探针与黄曲霉之间有微弱非特异性反应，但是不影响鉴定结果。45例临床真菌分离株的DNA微阵列鉴定结果与临床检出结果完全一致。结论该研究建立的DNA微阵列技术可在8h内快速、准确地检测临床常见深部感染真菌菌种，具备应用于临床辅助诊断的前景。Objective To develop a oligonucleotide microarray for detection of eight common deep fungi. Methods Eight clinical common deep fungi were choosen for our research,including Candida albicans,Candida glabrata,Candida krusei, Candida Ntropicalis, Candida parapsilosis, Aspergillus terreus, Aspergillus flavus, Aspergillus funigatus and Rhizopus oryzae. The ITS region of fungi was amplified with a pair of universal primers and labled with biotin in the meantime. The probes were designed in the variable region of target DNA sequence and fixed on the nylon membrane. Our DNA microarray was validated by using 45 clinical isolates as blinded samples. Results Eight clinical common deep fungi were detected with our DNA microarray successfully within 8 hours. The probes for Candida albicans,Candida glabrata ,Candida krusei, Candida tropicalis ,Candida parapsilosis ,Aspergillus terreus, Aspergillus flavus and Rhizopus oryzae had high specificity; The probes for Aspergillus funigatus had weak cross reaction with AspergiUus flavus,but it didn＇t influence the results of identification. The identification results of 45 clinical isolates by using our DNA microarray were consistent with clinical results. Conclusion Membrane-based microarray coupled with ITS probes possessed sufficient power to identify clinical common deep fungi rapidly and accurately in 8 hours and had the prospect of clinical applications.

关 键 词：常见深部真菌 DNA微阵列 核糖体RNA基因 

分 类 号：R379[医药卫生—病原生物学] R446[医药卫生—基础医学]