渝西地区奶牛隐孢子虫的分离和基因型鉴定

摘要

To identify Cryptosporidium species and genotypes in Western Chongqing, 18S rRNA genes were amplified by nested PCR and the products were analyzed by RFLP. The amplified fragments were cloned into pEGM-T easy vector and sequenced. The homology and phylogeny analysis of these sequences were conducted by using BLAST and MEGA 4. 0 softwares. The results indicated that the approximate 830 bp fragments of 18S rRNA gene were successfully amplified, and the PCR product was digested with restriction enzymes Ssp Ⅰ and Mbo Ⅱ , respectively. All the digested products were analyzed with RFLP assay. The results revealed the presence of all the three major Cryptosporidium species of dairy calves viz. , C. andersoni, C. ryanae and C. bovis. C. andersoni is the dominant species in Western Chongqing. This work will provide a valuable resource for further research on pathogeny biology of cryptosporidiosis and monitor this disease in Chongqing.%为鉴定渝西地区奶牛隐孢子虫分离株的种类和基因型,本研究利用巢氏PCR(Nested PCR)扩增18SrRNA基因,PCR产物进行限制性片段长度多态性分析(Restriction fragment length polymorphism analysis,RFLP)；将目的片段克隆至pGEM-T Easy载体,对阳性克隆进行测序,序列用BLAST和MEGA 4.0进行同源性和种系发育分析.结果表明阳性样品均成功扩增出约830 bp的目的基因片段,PCR产物分别经SspⅠ和MboⅡ酶切后进行RFLP分析,显示渝西地区存在3种隐孢子虫,即C.andersoni、C.bovis、C.ryanae.其中C.andersoni为优势虫种.鉴定结果为进一步开展奶牛隐孢子虫的病原生物学研究及该地区隐孢子虫病的监测奠定了基础.