PCR技术在住肉孢子虫虫种鉴定上的应用

Application of PCR to Identification of Sarcocystis Species

采用 RAPD 方法对水牛梭形住内孢子虫、待定种及黄牛枯氏住内孢子虫缓殖子基因组 DNA 进行了分析,待定种 PCR 产物的电泳带型与枯氏住肉孢子虫的差异较梭形住内孢子虫大,这与二者表型性状一致的研究结果不吻合.对3种包囊种特异性的 PCR 指纹进行了筛选,回收和纯化了3种包囊种特异性的 DNA 片段,待定种0.7kb 片段经α-^(32)PdATP 标记后用作探针,只与同源 DNA 杂交,不与其它包囊、宿主细胞和艾美耳球虫杂交,用 pGEM-T Easy Vector 对该片段克隆成功,并测出其全长序列,为进一步设计特异引物建立标准 PCR 诊断方法和用作核酸探针奠定了基础。

The genomic DNA of S.sp and S.fusiformis of buffalo,and S.cruzi of cattle were analysed by RAPD.Random amplified polymorphic DNA mobility patterns of S.sp of buffalo were more different with those of S.cruzi than with those of S.fusiformis.It is contradictious to similarity of phenotype between S.sp.and S.cruzi.Several arbitrary primers were used with DNA of three species of Sarcocystis to generate unique DNA fin- gerprints for differentiating each parasite and host DNA.Species-specific PCR products were obtained and purified.When the 0.7 kb DNA fragment was used as a DNA probe,it only hybridized with DNA in the microcysts of buffalo,not with DNA of the other two Sarcocystis species,hosts and Eimeria tenella.We have successfully cloned and determined the nueleotide sequence of this fragment with pGEM-T Easy Vector and Sanger's method to further characterize it for use as a probe and to design special primers in a standard PCR assay.