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目的克隆一例临床包虫病标本的线粒体基因COI、ND1和12S rRNA的基因序列,在基因水平上对包虫病病原体进行诊断,分析棘球绦虫线粒体基因序列差异。方法根据Gen Bank中登录的棘球绦虫线粒体基因COI、NDI和12S rRNA的序列信息分别设计3对引物,扩增临床包虫病标本的COI、NDI和12S rRNA基因,并进行测序。对所克隆的目的基因序列进行BLAST分析并与Gen Bank中已收录的部分棘球绦虫的COI、NDI和12S rRNA基因序列进行对比分析,并制做系统发生树。结果从包虫囊液DNA中成功克隆了长度分别为528 bp、1 001 bp和246 bp的基因片段,序列分析表明所克隆的基因片段为多房棘球绦虫的COI、NDI和12S rRNA基因。其中基因COI和NDI可以用于棘球绦虫的种间分类,而基因片段12S rRNA不能用于棘球绦虫的种间分类。结论从基因水平上确诊了此例包虫病患者的病原体为多房棘球蚴。
Objective To clone the gene sequences of mitochondrial COI, ND1 and 12S rRNA in a clinical echinococcosis specimen and to diagnose the echinococcosis pathogens at the genetic level and analyze the mitochondrial DNA sequence differences of the echinococcosis. Methods According to the sequence information of mitochondrial DNA COI, NDI and 12S rRNA registered in Gen Bank, three pairs of primers were designed respectively to amplify COI, NDI and 12S rRNA genes of clinical echinococcosis specimens and sequenced. BLAST analysis of the cloned target gene sequences and comparison with the COI, NDI and 12S rRNA gene sequences of the part of the genomic Echinococcus from GenBank, and the phylogenetic tree were made. Results The fragments of 528 bp, 1 001 bp and 246 bp were successfully cloned from hydatid cyst DNA. The sequence analysis showed that the cloned gene was COI, NDI and 12S rRNA gene of Echinococcus multilocularis. Among them, the gene COI and NDI can be used for the interspecific classification of Echinococcuss, while the gene fragment 12S rRNA can not be used for the interspecific classification of Echinococcuss. Conclusions The etiological agent of this case was diagnosed as Echinococcus multilocularis at the gene level.