羊泰勒虫病二温式PCR诊断方法的建立及初步应用

Establishment and Primary Application of Two-temperature PCR Method for Detection Theileriosis in goats and sheep

为建立特异、敏感、快速的羊泰勒虫病诊断方法。根据Gen Bank已报道的羊泰勒虫表面蛋白基因(AY274329)设计合成了1对引物,通过条件优化,建立了羊泰勒虫病二温式PCR诊断方法。该方法能扩增出335 bp的羊泰勒虫特异性基因片段,测序结果与已知基因序列同源性为100%。对羊泰勒虫基因组DNA的最小检测量为16 fg/μL,与新孢子虫、弓形虫、巴贝斯虫和瑟氏泰勒虫均无交叉反应。与普通PCR相比,二温式-PCR在敏感性方面无显著差异,但其反复升温降温时间消耗短。结果表明,二温式PCR诊断方法具有较高的特异性和敏感性,可用于羊泰勒虫病的快速诊断和流行病学调查。

To establish a rapid, specific and sensitive method for the detection of Theileriosis in goats and sheep, a pair of specific primers was designed and synthesized according to Theileria sp surface protein （SP） gene in GenBank （ AY274329 ） and a two -temperature PCR assay was established. A 335 bp gene fragment was amplified, and had 100% of homology with the known gene sequence. The minimum detectable amount of DNA was 16 fg/μL. The method showed no cross reaction with Neoapora caninum, Toxoplasma gondii RH strain, Babesia gibsoni and Theileria sergenti. Compared with common PCR,two -temperature PCR had no significant difference in sensitivity. But, the method took a short time with repeated heating and cooling. The results showed that the PCR method was specific and sensitive. This study provided a method for rapid clinical diagnosis and epidemiological investigation of Theileriosis in goats and sheep.