青海省2例锡兰钩虫的PCR-RFLP鉴定

PCR-RFLP identification of two cases of Ancylostoma ceylanicum in Qinghai Province

目的为了解青海省犬科动物流行的钩虫种类,为青藏高原地区钩虫的流行分布及种群进化研究提供依据。方法2014—2016年在青海省达日县和兴海县分别收集犬科动物粪便样本,洗脱获取粪便表面动物细胞,通过PCR扩增和测序对粪便标本溯源,以饱和蔗糖水漂浮法进行粪便虫卵检查,收集虫卵提取DNA,以聚合酶链反应-限制性内切酶片段长度多态性(PCR-restriction fragment length polymorphism,PCR-RFLP)法进行虫种鉴定。结果通过动物粪便溯源试验,获得动物线粒体D-loop区域长度为372bp的基因片段,运用基本局部比对搜索工具(basic local alignment search tool,BLAST)分析显示达日县和兴海县动物粪便标本来源分别为家犬和红狐;虫卵内转录间隔区(internal transcribed spacer,ITS)的PCR扩增,获得长度约为544bp的特异性电泳条带,PCR产物经限制性内切酶RsaⅠ和HinfⅠ的酶切反应显示特异性的酶切、电泳条带,两个钩虫样本均被鉴定为锡兰钩虫。结论锡兰钩虫首次在青藏高原地区报道,并在野生红狐体内被发现,有一定的生物学意义。

Objective To understand the hookworm species prevalent in canine animals in Qinghai Province,and to provide a basis for the study of the prevalent distribution and population evolution of hookworms in the Qinghai-Tibet Plateau Region.Methods From 2014 to 2016,canine fecal samples were collected in Dari and Xinghai counties of Qinghai Province,respectively.Animal cells on the surface of feces were obtained by elution.Fecal specimens were traced by PCR amplification and sequencing.Eggs in feces were examined using the saturated sucrose solution method,and recovered for DNA extraction.PCR-restriction fragment length polymorphism(PCR-RFLP)method was applied to identify the hookworm species.Results Through the traceability test for animal feces,a gene fragment with a length of 372bp was obtained from the mitochondrial D-loop region of animals.Under the application of basic local alignment search tool(BLAST),the analysis showed that the sources of animal feces specimens collected in Dari and Xinghai counties were identified as domestic dog and red fox,respectively.The PCR amplification of the internal transcribed spacer(ITS)region of the hookworm eggs yielded specific bands with a length of around 544bp observed in electrophoresis assay.The PCR products were applied to endonuclease digestion with specific enzyme RsaⅠand HinfⅠ,which revealed specific digestion and electrophoretic bands.Both hookworm samples were finally identified as Ancylostoma ceylanicum.Conclusion Ancylostoma ceylanicum is first reported in the Qinghai-Tibet Plateau Region and is found in wild red fox with some biological significance.