PCR-RFLP在细粒棘球绦虫种株鉴定中的应用

Appllication of PCR-RFLP method for identification of Echinococcus granulosus genotypes

目的根据细粒棘球绦虫线粒体DNA rrnL区段基因特征,建立一种新的细粒棘球绦虫种株(基因型)鉴定技术。方法应用PCR-RFLP方法,对新疆不同地区44个囊型包虫病(CE)病人分离株标本提取DNA,用特异性引物对mtDNA rrnL基因片段进行PCR扩增,将扩增产物用限制性内切酶SspI和Bg1Ⅱ消化,再用琼脂糖凝胶电泳分析片段大小,进行基因型鉴别。结果CE病人分离株PCR扩增产物均不能被限制性内切酶SspI酶切,为细粒棘球绦虫;其中43个病人分离株的PCR扩增产物不能被限制性内切酶Bg1Ⅱ酶切,为细粒棘球绦虫G1基因型;1个病人分离株的PCR扩增产物被Bg1Ⅱ切成158 bp和403 bp 2条DNA片段,为细粒棘球绦虫G6基因型。结论根据细粒棘球绦虫线粒体DNA rrnL区段基因特征建立的PCR-RFLP技术可用于细粒棘球绦虫的基因型鉴别。

Objective To establish a new mathod for verifying the typical strains of Echinococcus granulosus and clarifying the geographical distribution of its genotypes in Xinjiang,based on mitocondrial DNA rrnL gene specific fragment. Methods This study adopted a polymerase chain reaction-based restriction fragment length polymorphism（PCR-RFLP） method to analyze pathological samples obtained from 44 patients with cystic echinococcosis（CE） in different areas of Xinjiang.With mtDNA rrnL gene as the special attractant,a trail to digest the mtDNA rrnL gene by restrictive enzymes SspⅠ and Bg1Ⅱ were carried out after PCR amplification of a specific region of this gene,and the restriction fragments were analyzed by agarose gel electrophoresis. Results The PCR amplicons obtained using the mtDNAs extracted from echinococcal lesions from all CE patients as templates could not be cut by the restrictive enzyme SspⅠ,were E.granulosus.fourty-three of these PCR amplicons could not be cut by the restrictive enzyme Bg1Ⅱ,were E.granulosus genotype 1（E.granulosus G1）.On the other hand,one of these PCR amplicon was cut into two DNA fragments with molecular sizes of 158 bp and 403 bp,respectively,was confirmed as E.granulosus genotype 6（E.granulosus G6）. Conclusion It was testified by the current experimenThese results showedt that the genotype verification of E.granulosus by PCR-RFLP method is completely consistent with the verification by DNA sequence analysis,and can be used for differentiation of the E.granulosus genotypes.