DNA重复序列鉴定陕西省利什曼原虫

Identification of Leishmania in Shaanxi Province based on repeated DNA sequence

提取陕西省韩城市犬源利什曼原虫分离株(1份)、病犬骨髓(1份)、患者骨髓(3份)和白蛉(2份)共7份样品的利什曼原虫DNA,PCR扩增DNA重复序列,扩增产物经毛细管电泳后进行双向测序。采用DNAstar Seq Man软件进行序列拼接,与国内外发表的DNA重复序列进行比对,分析同源性,并用邻接法(NJ)构建系统进化树。结果 7份利什曼原虫DNA样品均扩增出260 bp左右的条带,与目的序列大小一致,且测序结果完全相同。序列比对结果显示,7条DNA重复序列与参考序列的婴儿利什曼原虫(GenBank登录号为L42486)、杜氏利什曼原虫(GenBank登录号为L42486)仅1个碱基的差异。同源性分析结果显示,同种原虫序列相似性大于90%,个别达100%。系统进化树结果显示,7条序列与杜氏利什曼原虫、婴儿利什曼原虫聚为一支。结合流行病学资料,陕西韩城利什曼原虫为婴儿利什曼原虫。

In this study, PCR was performed to amplify repeated DNA sequence in Leishmania DNA extracted from a sample of Leishmania isolate from dog, a bone marrow sample from an infected dog, 3 bone marrow samples from patients, and two Phlebotomus samples. The amplified products underwent bi-directional sequencing after capillary electrophoresis. The DNAstar Seq Man software was used for sequence assembly, and the sequences were aligned with other DNA repeated sequences reported in literature. Homology was analyzed and phylogenetic trees were constructed using the neighbor joining method. PCR resulted in a product of -260 bp for all the 7 samples,which was consistent with expectation and verified by sequencing. Results of sequence alignment showed that the products from the 7 samples differed from the reference sequences of L. infantum（Gen Bank No. L42486） and L.donovani（Gen Bank No. L42486） in only 1 base. Homology analysis revealed over 90% species homology to the ref-erence sequences, with some samples reaching 100%. The phylogenetic tree showed that the 7 sequences clustered into a branch with L. infantum and L. donovani. Combined with the epidemiological data, these results suggest that the Leishmania species in Shaanxi Province is L. infantum.