马尔尼菲青霉菌DNA检测及序列分析

Penicillium marneffei DNA Sequencing and Analysis

目的设计马尔尼菲青霉菌种特异性引物,探讨马尔尼菲青霉病早期诊断的方法。方法采用真菌通用引物ITS1和ITS4 PCR扩增我科保存的2株和广西医科大学附一院惠赠的1株马尔尼菲青霉菌rDNA ITS,扩增产物纯化后直接测序,测序结果在国际基因库核酸序列数据库进行同源序列搜索、比对、分析。结果3株临床分离的马尔尼菲青霉菌rDNA ITS序列相同;与来源于美国、印度尼西亚、法国、澳大利亚的马尔尼菲青霉菌的rDNA ITS序列一致;马尔尼菲青霉菌与荚膜组织胞浆菌、新生隐球菌、念珠菌以及曲霉和青霉属间的rDNA ITS序列差异较大,而青霉种间的rDNA ITS序列差异不大。结论不同来源的马尔尼菲青霉菌种间的rDNA ITS序列具有高度的同源性;提示该区不适合作为靶基因来设计该菌的种特异性引物或探针,应考虑在别的区域继续寻找。

OBJECTIVE To design the specific primers of Penicillium marneffei and to discuss the earlier-diagnosis method for penicilliosis marneffei. METHODS Three P. marneffei strains were clinically isolated, one gifted from the First Affiliated Hospital of Jianxi Medical College, the other two strains reserved in our department. The rDNA ITS of three strains was amplified by PCR with fungal general primers （ITS1 and ITS4）. The DNA sequence of the purified product was analyzed respectively and compared with the GenBank. RESULTS The rDNA ITS squence of the three isolates was the same, and showed no difference with the isolates from the USA, Indonesia, France and Australia, but showed a significant difference with the Histoplasma capsulatum, Cryptococcus neoforrnans, Candlda, and Aspergillus, and a little difference within the Penicilliurn species. CONCLUSIONS The rDNA sequence of P. marneffei strains from different region is highly homologous, this indicated the gene site is not suitable for designing specific primers or probe which could be used for different species, other sites should be explored.