摘要
旨在鉴定由海豚粪便样本中所分离所得线虫种类.首先对所分离虫体进行形态观察,然后提取虫体基因组DNA,并对样本28S rRNA D2-D3基因和ITS基因片段进行PCR扩增,以进行分子生物学鉴定.所得序列经BLAST比对并用邻接法构建系统进化树对所得序列进行分析.结果显示:虫体长120～140 mm,整体呈圆柱形,两端尖细,外观呈乳白色半透明,镜下观察其内部偏黄.可见食道前端细,向后逐渐膨大.PCR结果显示,28S rRNA D2-D3基因与ITS基因扩增长度分别为797和840 bp.BLAST比对结果显示:28S rRNA D2-D3基因(GenBank登录号OR345165)与已报道典型异尖线虫(Anisakis typical,GenBank登录号HF911524.1)的序列一致性为95.36％;ITS基因(GenBank登录号OR345164)与典型异尖线虫(GenBank登录号MF668919.1)一致性为97.11％,其中,ITS-1区域与典型异尖线虫(GenBank登录号MF668919.1)一致性为91.90％,ITS-2区域与典型异尖线虫(GenBank登录号JN968938.1)一致性为94.97％,5.8S rRNA区域与典型异尖线虫(GenBank登录号OQ244221.1)一致性为100.00％.基于ITS基因和28S rRNA D2-D3基因的系统发育分析结果显示,此次研究分离所得寄生虫样本与典型异尖线虫遗传距离最近,且同一分支上.本次分离所得寄生虫样本经过分子鉴定为典型异尖线虫.同源性分析结果显示,其与典型异尖线虫一致性均大于95％;同时,在28S rRNA D2-D3基因序列和ITS基因序列的系统发育分析方面,均与已报道的典型异尖线虫遗传距离最近.
Abstract
In order to identify the species of nematodes isolated from dolphin fecal samples,the morphology of the isolated nematodes is first identified.The parasite's body length was observed to be 120-140 mm long,with a cylindrical shape and slender ends.Its appearance was milky white and translucent,and its internal structure was observed to be yellowish under the micro-scope.It can be seen that the front end of the esophagus is thin and gradually expands back-wards.We extracted the genomic DNA of the isolated worm and amplified the 28S rRNA D2-D3 gene and ITS gene fragments by PCR for molecular identification.The PCR results showed that the amplification products of the 28S rRNA D2-D3 gene and ITS gene were 797 and 840 bp,re-spectively.BLAST alignment results showed that the sequence similarity between the 28S rRNA D2-D3 gene(GenBank accession number OR345165)and the Anisakis typical(GenBank acces-sion number HF911524.1)was 95.36％.The sequence similarity between the ITS gene(Gen-Bank accession number OR345164)and the Anisakis typical(GenBank accession number MF668919.1)was 97.11％,with a 91.90％sequence identity between the ITS-1 region and the Anisakis typical(GenBank accession number MF668919.1).There is a 94.97％sequence identi-ty between the ITS-2 region and the Anisakis typical(GenBank accession number JN968938.1)and a 100.00％sequence identity between the 5.8S rRNA region and the Anisakis typical(Gen-Bank accession number OQ244221.1).Phylogenetic analyses based on the ITS gene and the 28S rRNA D2-D3 gene showed that the parasite samples isolated in this study were genetically closest to the Anisakis typical,and on the same branch.In summary,the isolated parasite samples ob-tained from this study should be the larvae of Anisakis typical based on microscopic examination and molecular analysis.
基金项目
安徽省自然科学基金青年项目(2008085QC136)
维普
万方数据