马尔尼菲青霉临床分离株的rDNA ITS序列分析

目的 为设计马尔尼菲青霉种特异性引物,探讨马尔尼菲青霉病更加确切的诊断方法.方法 实验菌株为北京大学真菌和真菌病研究中心保存的4株马尔尼菲青霉,来源于国内不同地区.用真菌通用引物ITS1和ITS4 PCR扩增马尔尼菲青霉rDNAITS,扩增产物纯化后直接测序,测序结果在基因库核酸序列数据库进行同源序列搜索,并依据序列对比、分析.结果 4株临床分离的马尔尼菲青霉的rDNA ITS序列相同.与国外来源于美国、印度尼西亚、法国、澳大利亚的马尔尼菲青霉rDNA ITS序列基本一致.马尔尼菲青霉与荚膜组织胞浆菌、新生隐球菌、念珠菌的rDNAITS序列差异较大,青霉和曲霉属间rDNAITS的序列相似性较低,而青霉种间rDNAITS序列的差异不大.结论 不同来源的马尔尼菲青霉菌株间乃至不同青霉的种间的rDNA ITS序列均具有较高的同源性,提示该区可能不适于作为靶基因来设计马尔尼菲青霉的种特异性引物或探针.

Sequence Analysis of rDNA ITS of Clinical Isolates of Penicillium marneffei

Objective To analyze the sequence of rDNA ITS of Penicillium marneffei for developing an accurate diagnosis method of P. marneffei. Methods Four strains of P.marneffei were collected from different areas in China. Fungal general primers (ITS1 and ITS4) were used to amplify the internal transcribed spacer of rDNA of P. marneffei. The PCR products were purified and sequenced directly. Then the sequences were analyzed by comparing with the gene bank. Results The results showed that the four strains of P. marneffei shared almost the same rDNA ITS sequence which was similar with the data from different areas, such as the United States, Indonesia, France and Australia. The rDNA ITS sequences of P. marneffei were quite differenct from Histoplasma capsulatum, Cryptococcus neoformans and Candida species. The rDNA ITS sequences of Penicillium and Aspergillus share lower similarities, while there was no obvious difference of rDNA ITS sequences among Penicillium species. Conclusions rDNA ITS sequences among P. marneffei strains from different areas and even among different Penicillium species are highly homogenous, which suggest that this region may not be suitable as the target gene for designing species-specific primers or probes.