猫锡兰钩虫rDNA ITS1的扩增及分子鉴定

Isolation and identification of feline hookworm based on ribosomal ITS1 sequence analysis

为了对从猫粪便样品中分离的钩虫虫卵进行种类鉴定,本研究提取该虫卵基因组DNA,根据核糖体内转录间区1(ITS1)的保守序列设计钩虫属引物,通过PCR扩增、克隆、测序、相关软件分析,建立系统进化树,从分子水平对其进行种类鉴定。结果表明扩增出404 bp DNA片段,测序结果显示该片段包括309 bp ITS1和95 bp的5.8S rDNA,与NCBI中登录的序列进行比对以及采用Clustal X和MEGA5.1软件分析确定该钩虫为锡兰钩虫。这是我国首次建立钩虫的分子鉴定方法,也是40年来再次发现猫源锡兰钩虫,为锡兰钩虫分子生物学和分子流行病学研究奠定了基础。

To identify the species of Ancylostoma sp. eggs isolated from feces of stray cat, the genomic DNA was extracted from feces sample and the first ribosomal internally transcribed spacer region （ITS1） was used as gene marker for species identification. In addition, a DNA fxagment of 404 bp was amplified by PCR with a pair of species-specific primers designed according to the ITS1 region and cloned for sequencing, which contained 309 bp of ITS1 sequence and 95 bp of 5.8S rDNA gene. The sequence alignment and phylogenetic tree analysis showed that the Ancylostoma sp. was A.ceylanicum, the results laid a foundation for the molecular biology and molecular epidemiology study of A. ceylanicum in China.