人源隐孢子虫分离株种类鉴定与种系发育关系分析

Identification and phylogenetic analysis of a Cryptosporidium isolate from a patient admitted in hospital

目的确定郑州某医院分离到1株隐孢子虫(PRHN)所属种/基因型。方法用PCR对该分离株18S rRNA、HSP70、Cpn60和AOX进行PCR扩增,并对扩增产物进行测序,扩增序列经用DNAstar和ClustalX与GenBank参考序列比对,用PAUP4.0构建种系发育进化树,以确定该分离株与其他隐孢子虫虫种之间的亲缘关系。结果通过18S rRNA、HSP70基因分析该分离株与Cryptosporidium parvum(C.parvum)鼠基因型同源性最高,分别为99.9%和99.2%,在种系发育树上,PRHN均与C.parvum鼠基因型亲缘关系最近;对Chaperponin 60(Cpn60)和Alternative oxidase(AOX)进行序列分析时,该分离株与本实验室分离到的猪隐孢子虫(C.suis)亲缘关系最近。结论该分离株为C.parvum鼠基因型。

To confirm the species and genotype of a Cryptosporidium isolate from a patient admitted to a hospital in Zhengzhou,the genes coding for the small subunit of the ribosomal RNA, 70 kDa heat shock protein（Hsp70）, chaperponin 60 （Cpn60） and alternative oxidase（AOX）were amplified and sequenced from this isolate of this Cryptospordium, and the amplified sequences were compared with the reference sequences in GenBank by DNA star and Clustal X softwares. The phylogenetic tree was established by analysis with the PAUP 4.0 software in order to reveal the inter-relationship of this isolate with other species of Cryptospordium. It was found that the sequence identities of this isolate with Cryptospordium parvum murine genotype appeared to be 9 9.9% and 99.2% ,the highest among all species of Cryptospordium,as demonstrated by 18S rRNA and Hsp70 genes analysis. The phylogenetic relationship of this isolate was proved to belong to C. parvum murine genotype clusters,but the results of DNA sequence analysis revealed that this isolate clustered most closely with the swine genotype of Cryptospordium suis isolates derived from pigs in Henan province.