基于线粒体COI、NDI和12S rRNA基因序列的包虫病标本的分子分类学研究

Molecular taxonomy of a hydatid disease specimen based on mitochondrial COI,NDI and 12S rRNA gene sequences

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 diagnose a case of hydatid disease at genetic level and to analyze the sequence diversities of mitochondrial genes of echinococcosis by cloning genes including COI,ND1 and 12 S rRNA. Methods Three pairs of primers were designed by analyzing some mitochondrial gene sequences in the Gen Bank database. Mitochondrial genes COI,NDI and 12 S rRNA of a hydatid specimen were amplified respectively using the 3 pairs of primers and their sequences were then sequenced.The cloned gene sequences were analyzed by BLAST and compared with the sequences of COI,NDI and 12 S rRNA of Echinococcus in Gen Bank,and phylogenetic trees were constructed. Results Three gene fragments with lengths of 528 bp,1 001 bp and 246 bp,respectively,were successfully cloned from the hydatid cyst fluid DNA. Sequence analysis showed that the cloned gene fragments were COI,NDI and 12 S rRNA genes of Echinococcus multilocularis. The cloned COI and NDI gene segments could be used for interspecies classification of Echinococcus,while the 12 S rRNA gene segment could not. Conclusion The causative agent of the clinical hydatid disease case was confirmed as Echinococcus multilocularis at genetic level.