套式PCR扩增山羊吕氏泰勒虫18S rRNA基因

泰勒虫是一种危害动物的重要血液寄生性原虫,常寄生于牛、羊、骆驼和其他野生动物,在中国的东北、西北等地较为流行。采集安徽定远县某山羊场疑似焦虫病的山羊血样,首先采用血涂片法检查,再用Blood DNA&Tissue kit提取血液基因组,参照Kim和Wei的方法设计2对引物用于泰勒虫18S rRNA基因的扩增,并对该方法进行敏感性和特异性分析。结果显示:血涂片检查明显可见红细胞内有典型的虫体,大小为0.6~2μm,初步怀疑为泰勒虫(Theileria);PCR扩增可扩增出359 bp大小目的片段,与预期结果相一致;基因序列同源性表明,该泰勒虫分离株基因与已报道泰勒虫毒株(JQ926740.1)核苷酸同源性最高,可达99.72%;在遗传进化方面,所分离的泰勒虫和吕氏泰勒虫(Theileria luwenshuni)Nanjingdong(JQ926740.1)最接近,而且在一个分支上。本试验所建立的套式PCR具有良好的特异性,且敏感性较高,最低测出量是3.8 fg/μL。本次分离的羊血液原虫为吕氏泰勒虫。

Amplification of the 18S rRNA gene of Theileria luwenshuni by nested PCR

Theileria, as a blood parasite, often infects cattle, sheep, camels and other wild animal.This parasitic potozoon is prevalent in the Northeast and Northwest in China.In this trial, blood samples were collected from goats on a farm in Dingyuan County, Anhui Province.The blood smear method was used to examine the samples, the Blood DNA &Tissue kit(QIAGEN) was used to extract genes from the blood samples, the methods reported by Lim and Wei was used to design two pairs of primers to amplify the 18S rRNA gene of Theilerias, and the sensitivity and specificity of the established PCR technique were analyzed.The results were that the blood smear examination showed typical Theileria in erythrocyte whose size was 0.6-2 μm;that PCR amplified the target gene to be 359 bp, as was consistent with the anticipated results;that comparison with the GenBank showed the target gene to be 99.72% in homology with the reported Theileria(JQ926740.1), which was closest to of Theileria luwenshuni Nanjingdog(JQ926740.1), of the same the same branch in the phylogenetic tree.The established nested PCR had a good specificity and high sensitivity, with minimum detectable quantity of 3.8 fg/μL could be detected.The protozoon isolated from the goat blood in this study was Theileria luwenshuni.