改进的多重PCR技术鉴定白色念珠菌、新生隐球菌及烟曲霉的实验研究

目的]建立一种快速而简便的鉴定白色念珠菌、新生隐球菌及烟曲霉的分子生物学方法。方法]应用通用引物ITS1与ITS4，PCR扩增真菌的内转录间隔区（包含5．8SrDNA），然后将白色念珠菌、新生隐球菌、烟曲霉的单条种特异性异物与引物ITS1结合，对通用引物的扩增产物进行多重PCR扩增，达到鉴定上述菌株的目的。结果]实验所采用的所有致病真菌，经通用引物的PCR扩增均能扩增出特异性的条带，而非真菌菌株扩增为阴性；白色念珠菌、新生隐球菌、烟曲霉的3种种特异性引物与ITS1结合进行的多重PCR扩增，只对目的菌株进行了有效扩增，其他菌株扩增为阴性。应用此方法对临床分离的菌株，白色念珠菌，新生隐球菌，烟曲霉进行鉴定，也得到特异性扩增条带。结论]改进的多重PCR技术，更加经济，简单，快速，为致病真菌菌种的鉴定提供了有效手段。

Identification of Candida albicans, Cryptococcus neoformans and AspergiUus fumigatus by modified multiplex PCR

Objective] To establish a rapid and simple molecular biological method for identification of Candida albicans, Cryptococcus neoformans and Aspergillusfumigatus. Methods] The contiguous ITS and 5.8SrDNA regions were amplified by using the universal primers ITS1 and ITS4. the amplified ITS products were amplified by multiplex PCR with the species - specific primers of the Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus combined with the primers ITS1. Results ] The specific fragment of pathogenic fungi in this study could be amplified by the universal primers ITS1 and ITS4, whereas non - fungi strains could not produce positive amplified fragments. Only Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus could produce positive amplified fragments by multiplex PCR with the species - specific primers of the Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus. The primer ITS1 and other strains could not produce positive amplified fragments. The clinically isolated strains of Candia albicans, Cryptococcus neoforroans and Aspergillusfumigatus could also produce positive amplified fragments by this method. Conclusion] Modified multiplex PCR is a simpler and more economic, rapid and powerful tool for identification of the pathogenic fungi.