Objective To establish a method for detecting and identifying clinically important fungi by PCR combined with oligonucleotide probe hybridization. Methods The DNA from 11 species of fungi (Candida albicans, Candida tropicalis, Candida pseudotropicalis, Candida parapsilosis, Candida glabrata, Candida liplytica, Candida guilliermondii, Candida krusei, Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus) were amplified with universal primers for 28s rRNA. The products were then identified by hybridization with 9 species-specific oligonucleotide probes labeled with biotin. The method was used to differentiate the clinical specimens and culture isolates. Results Universal primers can amplify 28s rRNA gene from 11 species of fungi. Each species-specific probe only hybridizes with its target molecules among 11 species of fungi. Dot hybridization has the same sensitivity as Southern hybridization. The results obtained by testing clinical specimens and culture isolates were quite specific. Conclusion The PCR method combined with oligonucleotide probe hybridization is proved to be a fast, sensitive and specific technology, and is potential for clinical application.%目的建立PCR结合生物素标记的寡核苷酸探针斑点杂交技术，鉴定临床常见的真菌。方法首先用真菌通用引物扩增白念珠菌、热带念珠菌、假热带念珠菌、近平滑念珠菌、光滑念珠菌、解脂念珠菌、克鲁斯念珠菌、季也蒙念珠菌、黄曲霉、烟曲霉的核糖体大亚单位基因的保守区序列，然后用生物素标记的种特异性寡核苷酸探针与扩增产物杂交。并将此方法用于临床标本和临床分离菌株的检测。结果通用引物可以扩增上述11种临床常见真菌的DNA，扩增片段长度在260bp左右。9种特异性探针分别与11种真菌标准菌株的PCR扩增产物杂交，结果表明每种探针都具有高度特异性。斑点杂交法和Southern杂交法检测敏感性相同，为100fg；琼脂糖凝胶电泳法检测敏感性为1pg。通过69例临床标本和31例临床分离菌株的检测，PCR-杂交法的结果和真菌培养法的结果基本一致。结论 PCR结合生物素标记的寡核苷酸探针杂交技术可将9种临床常见真菌鉴定到种，方法快速、敏感、特异。
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