犬复孔绦虫ITS及5.8 S rDNA的PCR扩增、克隆及序列分析

PCR Amplification,Cloning and Sequence Analysis of the ITS and 5.8 S rDNA of Dipylidium caninum

目的以从我国广东广州和湛江犬小肠中采集的2条犬复孔绦虫作为研究对象,以保守引物NC5及NC2扩增犬复孔绦虫的ITS-1,5.8 S及ITS-2 rDNA片段并进行序列分析。方法将PCR扩增出的片段纯化后克隆至pGEM-T Easy载体,重组质粒通过菌落PCR和酶切鉴定后,对阳性菌落进行序列测定。结果来自广州和湛江的2条犬复孔绦虫ITS及5.8 S rDNA序列总长分别为1536bp、1385bp,2条犬复孔绦虫的ITS-1、ITS-2序列相差较大,分别为20.80%、27.17%,而5.8 S序列相差较小(1.49%)。结论由于犬复孔绦虫ITS序列复杂,种内存在的差异大,故不适于作为犬复孔绦虫种的遗传标记。

Objective To amplify and analyse the internal transcribed spacer （ITS） and 5.8 S rDNA of Dipylidium caninum isolated from Guangdong province. Method The ITS and 5.8 S rDNA of D.caninum was amplified by PCR using a pair of conserved primers and the amphcons were cloned into pGEM-T Easy vector. Result The inserts were successfully sequenced,and the results revealed that the ITS of D.canintan sample DcGZ was 1536 bp,and the sample DcZJ was 1385 bp in length.Sequence analysis revealed that the ITS-1 and ITS-2 of the two samples differed significantly,and the sequence difference were 20.80% and 27.17% respectively,but there was only a slight difference in the 5.8 S sequence between the two samples. Conclusion The results of the present study showed that the ITS sequence was not suitable as geneti marker of D.caninum.