两种念珠菌DNA探针的研制及应用

目的：制备两种念珠菌DNA探针并应用于临床诊断。方法：对聚合酶链反应扩增的白色念珠菌标准株的E03基因的125bp片段，用半抗原地高辛标记；合成仪合成的E03基因中25bp特异寡核苷酸，用生物素标记。检测两种探针显色敏感度，并与23株临床分离的白色念珠菌、热带念珠菌，近平滑念珠菌、克柔念珠菌、星形念珠菌及大肠埃希菌等细菌进行斑点杂交试验。结果：Dig-125bpDNA探针显色敏感度为0.01pg，仅与白色念珠菌杂交，且对临床分离株检测的结果与传统厚膜孢子鉴定法相符。Bio-25bpDNA探针显色敏感度为20pg，与白色念珠菌、近平滑念珠菌及星形念珠菌杂交。结论：Bio-25bp DNA探针可用于念珠菌初步鉴定，而Dig-125bp DNA探针可用于白色念珠菌的鉴定。

Preparation and using of two kinds of Candida DNA probe

Objective Preparation and using of Candida DNA probe. Methods The 125 hp DNA from E03 gene of C. albicans was amplified by PCR, then by hepen digoxigenin, and the 25 bp specific sequence from E03 gene was synthesized and labeled with biotin. The sensitivity of these probes was detected, and the specificity was detected by dot-blotting hybridization assay with 23 strains C. albicans isolated from patients, C. paropsilosis, C. tropicalis, C. krusei, C. stellatoidea and E. coli and so on. Results The Dig-125 hp DNA probe, only hybridized with C. albicans, whose sensitivity of color development was 0. 01 pg, and the hybridization result of the isolated strains was coincidental with the result of traditional identification for C. albicans. The Bio-25 hp DNA probe, hybridized with C. albicans, C. paropsilosis, C. tropicalis and C. stellatoidea, whose sensitivity of color development was 20 pg. Conclusion The Dig-125 bp DNA probe is specific and sensitive to C. albicans and suitable for the indentification of clinical C. albicans infection, while the Bio-25 bp DNA probe is suitable for the indentification of clinical candida infection.