文献类型：期刊文献

中文题名：18Sr RNA基因巢式PCR-RFLP分析两株牛源隐孢子虫分离株

英文题名：Isolation of two strains of Cryptosporidium from bovines by 18S rRNA gene nested PCR-RFLP

作者：刘晖[1,2];袁忠英[2];沈玉娟[2];冯耀宇[3];曹建平[2]

机构：[1]遵义医学院寄生虫学教研室,遵义563003;[2]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织疟疾,血吸虫病和丝虫病合作中心,上海200025;[3]华东理工大学资源与环境工程学院,上海200237

年份：2009

卷号：25

期号：2

起止页码：115

中文期刊名：中国人兽共患病学报

外文期刊名：Chinese Journal of Zoonoses

收录：CSTPCD;;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；

基金：卫生行业科研专项(No.200802012);国家"十五"科技攻关项目(No.2003BA712A03-06)联合资助

语种：中文

中文关键词：牛隐孢子虫;安氏隐孢子虫;18S;rRNA基因;巢式PCR—RFLP

外文关键词：Cryptosperidium boris; Cryptosperidium andersoni;18S rRNA gene;nested PCR-RFLP

摘要：目的18S rRNA巢式聚合酶链反应(Nested PCR)-限制片段长度多态性(restriction fragment length polymor-phism,RFLP)鉴定上海(简称SH-BOV)和徐州(简称XZ-BOV)两株牛源隐孢子虫。方法改良-抗酸染色确定感染隐孢子虫的上海株和徐州株牛粪,提取DNA后经18S rRNA基因巢式PCR扩增,扩增产物测序后用Blast和MEGA软件进行同源性和系统发育分析。同时扩增产物分别用SspⅠ和VspⅠ单酶切,且XZ-BOV扩增产物用DdeⅠ酶切后进行RFLP分析。结果通过18S rRNA基因分析,SH-BOV与1株巴西牛隐孢子虫Cryptospridiumbovis同源性为100%,种系发育树上两者在同一分支;XZ-BOV与安氏隐孢子虫C.andersoni同源性为99%,种系发育树上两者在同一分支。扩增产物分别用SspⅠ和VspⅠ单酶切后,可鉴别出SH-BOV为C.bovis,XZ-BOV为C.andersoni或C.muris。XZ-BOV扩增产物用DdeⅠ酶切后可鉴别出XZ-BOV为C.andersoni。结论上海株牛源隐孢子虫(SH-BOV)鉴定为牛隐孢子虫(C.bovis),徐州株牛源隐孢子虫(XZ-BOV)鉴定为安氏隐孢子虫(C.andersoni)。
Two strains of Cryptosporidiurn were isolated from feces of naturally infected bovines in Shanghai （SHBOV） and Xuzhou （XZ-BOV） were identified by 18S rRNA gene nested PCR-RFLP. The genomic DNA of Cryptosporidium identified by the modified acid-fast staining was extracted and amplified by using the 18S rRNA gene nested PCR-RFLP assay, and Blast and MEGA softwares were used to analyze their homology and phylogeny. Meanwhile, their amplified products were digested with restriction enzymes Ssp I and Vsp I, while those of XZ-BOV also digested with Dde I enzyme. All these digested products were analyzed with RFLP assay. As demonstrated by 18S rRNA gene analysis, the homology of SH-BOV strain and C. boris isolated from Brazil was 100%, and they situated at the same branch of the phylogenic tree, while that of XZ-BOV strain and C. anderson was 99%, but they were also at the same branch of phylogenic tree. After the amplified products were digested respectively with Ssp f and Vsp I restriction enzymes, SH-BOV strain was identified as C. boris, while XZ-BOV strain identified as C. andersoni or C. muris. In additioin, when XZ- BOV strain was digested with Dde I enzyme, it was identified as C. andersoni. It is concluded that the SH-BOV strain isolated from Shanghai is C. boris, whereas the XZ-BOV strain isolated from Xuzhou is C. andersoni.