犬源环孢子虫的ITS-1序列鉴定

Molecular identification of dog-derivedCyclospora sp. based on ITS-1 sequence

目的对犬粪中新发现的环孢子虫进行分子鉴定。方法在GenBank中登录的环孢子虫18S和5.8S rDNA序列的保守区设计一对引物ITS-1+F/ITS-1+B,扩增犬源环孢子虫ITS-1+序列;将扩增产物纯化并连接到质粒载体pMD19-T,克隆转入感受态细胞JM109,挑选阳性克隆测序并进行相似性比较和种类鉴定。结果犬源环孢子虫ITS-1+序列长为547bp,包含58bp的18S rDNA序列,333bp的ITS-1序列和156bp的5.8S rDNA序列。其中的18S rDNA和5.8S rDNA与艾美耳科原虫的相似性最大;其中的ITS-1序列与人源环孢子虫、牛源环孢子虫、狒狒源环孢子虫均不相同。结论该犬源环孢子虫可能为环孢子虫属的一个新种。

To identify one Cyalospora sp.from dog feces,a pair of primers ITS-1＋F/ITS-1＋B were designed to amplify the ITS-1＋ region of the Cyclospora-like species from dog by PCR amplification based on the published sequence of the rDNA gene of Cyclospora in GenBank.The amplicons were purified and cloned into pMD-19T vector followed by being transfected into JM109.The positive clones were cultured and identified by PCR amplification and the insert was successfully sequenced.The result revealed that the amplicon was 547bp in length,including 58bp of 18S rDNA,333bp of ITS-1 and 156bp of 5.8S rDNA.The dog associated Cyclospora-like organism belongs to Eimeriidae based on the high similarity of its partial 18S rDNA and 5.8S rDNA and other protozoans of Eimeriidae.The organism might be a new species based on its specific ITS-1 sequence.