摘要
目的鉴定1株由脑膜炎患者分离的无绿藻菌株。方法常规的形态学、生化方法及扩增相关基因序列进行菌种鉴定。选用真菌通用引物扩增核糖RNA(rRNA)28S大亚基(LSU)、核糖体内转录间区(ITS) DNA;真核细胞特异性引物及无绿藻种属特异性引物扩增核糖体18S小亚基(SSU)DNA。序列结果用BLAST软件分析(网址:www.ncbi .nl m.nih.gov/BLAST)。比对一致性在99 %以上的菌株,作为正确的鉴定结果。结果该菌在25℃、37℃均可生长,沙堡弱培养基(SDA)、马铃薯培养基(PDA)见光滑湿润的白色酵母样菌落,镜下圆形、椭圆形的孢子及裂殖成圆形或不规则形的内孢子,无菌丝、子囊、芽孢,根据形态学特征鉴定为无绿藻。API 20C AUX鉴定系统提示与威克汉姆无绿藻的一致性为2 .9 %。核酸序列分析显示核糖体内转录间区( ITS) DNA可能存在复杂结构,不适合无绿藻的测序鉴定;但核糖体大亚基(LSU)及小亚基(SSU)DNA序列分析,与祖菲无绿藻碳水化合物变种(Prototheca zopfii var . hydrocarbonea)一致性分别为94 %、99 .9 %。结论形态学是菌种鉴定基础,形态学结合核酸序列分析的方法,可使该临床罕见菌株,鉴定至种的水平。在所选择的基因序列中,18S SSU rDNA比28S LSU rDNA序列分析,更有利于该临床分离株的鉴定。
A strain of Prototheca species isolated from a case of meningitis was identified by routine morphologic and biochemical methods as well as amplification of the related genes, in which the 28S large-subunit (LSU) region of ribosomal RNA (rRNA) gene and intergenic space (ITS) were amplified with universal fungal primers. The small-subunit (SSU) rRNA gene was amplified with eukaryote-specific primers and Prototheca genus-specific primers. Then, compared the sequences with the ones posted on BLAST (www. ncbi. nlm. nih. gov/BLAST). The organism choice giving the closest match, up to 99%, was considered the most likely correct identification. It was found that this strain of fungus grew well at 25 ℃ or 37 ℃. Smooth, moist colonies with white color were observed on Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA). Microscopically, globular or ovoid cells, a number of round, ovoid shaped endospores could be observed. No hypha, ascus or blastic conidia was found upon cultivation on SDA. Based on the morphological characteristics, this isolate could be identified as Prototheca species. The identity with Prototheca wickerhamii was 2.9% as demonstrated by the API 20℃ AUX system. Sequence analysis showed that the ITS gene was proved to be a complex structural region which was not suitable for the identification of Prototheca species, but the LSU and SSU rDNA regions showed 94% and 99.9% sequence identities with Prototheca zopfii var. hydrocarbonea (P. zopfii var. hydrocarbonea) respectively, indicating that the SSU rRNA gene sequence might be more reliable on than the LSU rRNA gene sequence for identification of Prototheca species.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2007年第7期656-660,共5页
Chinese Journal of Zoonoses
基金
This work was supported by Reform Human Resource Management Foundation(code:00-722) of China Medical Board of New York Inc
关键词
祖菲无绿藻碳水化合物变种
测序
鉴定
Prototheca zo p f ii var. h ydrocarbonea
identification
sequence