山西省及周边地区利什曼原虫SSU rRNA基因和ITS-1序列克隆及序列系统进化分析

CLONING AND PHYLOGENETIC ANALYSIS OF SSU RRNA AND ITS-1 SEQUENCES OF LEISHMANIA IN SHANXI PROVINCE AND ITS SURROUNDING AREAS

为了解山西及周边地区内脏利什曼病的病原体利什曼原虫(Leishmania spp.)的虫种类型及其基因多态性,使用首都医科大学附属北京友谊医院收治的13例来自山西及周边地区的内脏利什曼病患者的骨髓样本提取DNA,分别扩增其核糖体小亚基rRNA(small subunit ribosomal RNA,SSU rRNA)和核糖体DNA内转录间隔区Ⅰ(ITS-1)并应用其序列构建系统发育树确定患者感染原虫的虫种类型及其遗传关系。结果显示,13个临床样本SSU rRNA和ITS-1序列同源性分别为99.59%和99.93%。基于SSU rRNA构建的系统发育树显示13个患者感染的虫种与杜氏利什曼原虫L.donovani、婴儿利什曼原虫L.infantum和恰氏利什曼原虫L.chagasi聚为一支。基于ITS-1构建的系统发育树显示13例患者感染的利什曼原虫均属于婴儿利什曼原虫。SSU rRNA与ITS-1序列分类的结果一致,ITS-1在利什曼原虫虫种鉴定上较SSU rRNA分辨率高,可鉴定到亚种水平。13例山西及周边地区利什曼病患者感染原虫均为婴儿利什曼原虫L.infantum,感染虫株间同源性较高。

To identify the species and molecular genetic characteristics of Leishmania protozoa in Shanxi Province and its surrounding areas.The SSU rRNA and ITS-1 sequences of Leishmania were amplified using DNA,extracted from bone marrow samples of patients with visceral leishmaniasis(VL)treated in Beijing Fridendship Hospital,Capital Medical University,sequence alignment were analyzed with the SSU rRNA gene and ITS-1 region sequences of Leishmania in different regions and species reported in the literature and the phylogenetic tree were constructed.The homology of SSU rRNA and ITS-1 sequences from 13 VL patients were 99.59% and 99.93% respectively.Phylogenetic tree analysis based on SSU rRNA gene showed that Leishmania from 13 VL patients were gathered into one cluster with L.donovani,L.infantum and L.chagasi.Phylogenetic tree analysis based on ITS-1 region sequences showed that Leishmania from 13 VL patients were gathered into one cluster with L.infantum.The results of SSU rRNA and ITS-1 sequences classification are consistent,ITS-1 has higher resolution than SSU rRNA in the identification of Leishmania species.SSU rRNA and ITS-1 sequences analysis showed that the pathogens of visceral leishmaniasis in Shaanxi Province and its surrounding areas are L.infantum.