应用长臂光敏生物素核酸探针检测医学真菌的研究  

The detection of medical fungi using DNA probe labelled with long arm photobiotin

作　　者：陈建魁[1] 尹秀云[1] 高亚兵[2] 佟雅丽[1] 李玉荣[1] 朱晓华[1] 彭瑞云[2] 牟兆钦[1] 

机构地区：[1]军事医学科学院附属307医院,北京100039 [2]军事医学科学院放射医学研究所,北京100850

出　　处：《中国卫生检验杂志》2003年第2期158-159,共2页Chinese Journal of Health Laboratory Technology

摘　　要：〔目的〕建立PCR结合长臂光敏生物素核酸探针杂交检测医学真菌的方法。〔方法〕首先合成医学真菌通用引物 ,用PCR扩增白色念珠菌 18SrRNA基因 ,用长臂光敏生物素标记纯化的扩增产物作为核酸探针 ,然后用此探针对PCR后的基因扩增产物进行Southern杂交检测医学真菌。通过用白色念珠菌感染人鼠 ,建立念珠菌病的实验动物模型 ,验证血培养法、PCR法和PCR扩增及Southern杂交检测医学重要真菌的敏感性。〔结果〕通用引物可以扩增白色念珠菌、热带念珠菌、新型隐球菌、黄曲霉菌、烟曲霉菌等 9种医学常见真菌的 18SrRNA基因 ,扩增片段长度为 62 1～ 687bp。此引物只扩增真菌DNA ,不扩增临床常见的细菌、病毒和哺乳动物组织细胞DNA。利用实验动物模型比较了血培养法、PCR法和PCR扩增及长臂光敏生物素核酸探针Southern杂交法检测真菌血症的灵敏度 ,实验证明后者的敏感性最高。〔结论〕本方法快速、敏感、不需放射性同位素 ,有较好的临床应用前景。睴bjective〕To develope a molecular method to detect the medical fungi using DNA probe Labelled with long arm photobiotin.〔Methods〕 At first, a pair of universal primers of medically important fungi were designed and synthesized. Then the DNA of Candida albicans were amplified with the universal primers. The amplification products were purified and labelled with the long arm photobiotin as a probe to be used in the Southern blot analysis of the medically important fungi. 〔Results〕 The 9 species of fungi including Candida albicans, Candida tropicalis, Candida kefyr, Candida parapsilosis, Candida glabrata, Candida lipolytica, Cryptococcus neoformans, Aspergillus fumigatus and Aspergillus flavus can be detected in the patients' samples. All the bacteria, virus and human cells tested give negative results. Comparing with the routine blood culture method and PCR method, the Southern blot method using probes labelled with Long arm photobiotin is more sensitive. It is able to detect the presence of as few as 5 cell or 10 fg DNA of fungus. 〔Conclusions〕It is a rapid, sensitive and safe method which does not use radioactivity. It has a good prospect of clinical application.

关 键 词：长臂光敏生物素 核酸探针 检测 医学真菌 白色念珠菌 动物模型 基因扩增产物 

分 类 号：R446.5[医药卫生—诊断学]