猴源环孢子虫的分离与分子鉴定

Isolation and molecular identification of Cyclospora-like organism from monkey

采用饱和蔗糖漂浮法、改良抗酸染色法和孢子化试验首次从广东某猴场的猴粪中分离到疑似环孢子虫卵囊。为了对其进行分子鉴定,采用巢式PCR和普通PCR方法,针对环孢子虫属18SrDNA和ITS基因设计了3对引物,扩增其目的片段,并进行了序列分析。结果显示,卵囊大小为7.5～10μm,经改良抗酸染色的卵囊染色程度不一,孢子化卵囊内含2个孢子囊,每个孢子囊内含有2个子孢子。通过PCR扩增,获得的18SrDNA目的片段长712bp,测序结果表明该猴源环孢子虫18SrDNA基因与Cyclosporacolobi、狒狒环孢子虫、人环孢子虫的相似率较高,在进化树上位于同一分支。获得的ITS-1+片段长760bp,其中ITS-1序列高度特异,在GenBank中未见同源性序列。结果表明,从猴粪中分离的疑似环孢子虫卵囊应属于环孢子虫。

Cyclospora-like oocysts from monkey feces were isolated for the first time in China by satu-rated sucrose floatation,modified acid-fast staining and sporulation experiment. To further identify the Cy-clospora-like organism, 18 S rDNA and ITS-1 were amplified by nested or conventional PCR with three pairs of primers designed according to related gene sequences of Cyclospora. The resulting amplicons were sequenced. The result revealed that the oocysts were spherical and they were 7.5 to 10 μm in diameter. Af-ter being stained with the modified acid-fast staining method, they appeared variance in color. There were two sporocysts in the sporulated oocyst, each containing two sporozoites. A 712 bp fragment of the 18 S rD-NA gene was amplified by PCR and shared higher similarity with that from Cyclospora colobi ,C. papionis and C. cayetanensis. The Cyclospora-like organism, Cyclospora colobi , C. papionis and C. cayetanensis were classified into the same clade in the phylogenetic tree. The amplified ITS-1＋ gene fragment was 760 bp in size and highly specific. It was concluded that the Cyclospora-like organism from monkey belonged to Cyclospora.