五步蛇源隐孢子虫分离株的分子鉴定

Molecular identification of Cryptosporidium isolate from a long-noded pit viper(Deinagkistrodon acutus)

【目的】采用分子生物学方法鉴定1个五步蛇(Deinagkistrodon acutus)源隐孢子虫分离株,了解中国蛇隐孢子虫感染的种类分布。【方法】利用巢式PCR方法,扩增隐孢子虫18S rRNA基因特异性片段,对PCR产物进行限制性片段长度多态性分析(PCR-RFLP)和同源性分析,构建其种系发育进化树。【结果】成功扩增出18S rRNA基因目的片段,PCR产物经SspⅠ酶消化后获得的酶切结果和先前报道的蛇隐孢子虫(Cryptosporidium serpentis)一致。获得的840 bp 18S rRNA基因序列与C.serpentis同源性达到99.8%。种系发育进化树分析表明,该分离株与C.serpentis位于同一分支,节点支持值达100%。【结论】本研究获得的五步蛇源隐孢子虫分离株为C.serpentis,说明C.serpentis有更宽的感染宿主范围。

【Objective】 The study was made to identify one Cryptosporidium isolate from a long-noded pit viper（Deinagkistrodon acutus） by molecular methods,thus to understand the distribution of Cryptopsoridium spp.in reptiles in China and to assess the public healthy significance.【Method】 A nested PCR method was used to amplify specific fragment of 18S rRNA gene in Cryptosporidium,PCR product was analysed by restriction fragment length polymorphism analysis（PCR-RFLP）,and phylogenetic relationship was calculated.【Result】 The expected fragment of 18S rRNA gene was successfully amplified,and the PCR product was digested and the profile of fragments was in agreement with that of Cryptosporidium serpentis（C.serpentis） reported previously.A 840 bp fragment of 18S rRNA gene was obtained by sequencing,and the similarity was 99.8% between this isolate and the C.serpentis.Among these Cryptosporidium species/genotypes shown in the phylogenetic ralationship tree,the present isolate formed the same taxon with C.serpentis,with the bootstrap value being 100%.【Conclusion】 The Cryptosporidium isolate from a long-noded pit viper was identified as C.serpentis.Thus,the result showed that the host range of C.serpentis was wider.