进境太平洋鳕鱼寄生的异尖科线虫rDNA-ITS 序列的PCR扩增及分析

以日本进境的冻太平洋鳕鱼体内分离出的异尖科线虫为研究对象,采用寄生虫通用引物NC5和NC2扩增其核糖体DNA(rDNA)的内转录间隔区(ITS)序列,进行克隆、转化、测序和序列分析,并对样品进行分子鉴定。结果表明,扩增的异尖科线虫样品的ITS序列片段大小为906 bp,包含部分的18S、28S及全部的ITS1(353 bp)、5.8S(157 bp)和ITS2(299 bp)序列,ITS1和ITS2序列与GenBank登录的伪新地蛔线虫(Pseudoterranova decipiens)同源性均为在99.7%以上,与其他线虫的相似性较低。由ITS1和ITS2序列构建的系统进化树可知,从鳕鱼中分离到的线虫ITS1和ITS2均与伪新地蛔线虫处于同一分支。本研究结果为异尖科线虫种属的确定及进一步的分子生物学研究奠定基础。

PCR Amplification and Sequence Analysis of ITS rDNA of Anisakid larvae in Imported Pacific Cod

The aims of this study were to amplify and analyze the sequence of internal transcribed spacers(ITS)of ribosomal DNA(rDNA)of Anisakid larvae samples isolated from frozen pacific cod imported from Japan.The DNA of the samples were extracted and the ITS sequences were amplified by PCR with primers NC5and NC2.The products were cloned into pMD18-T vector and the inserts were successfully sequenced.The results revealed ITS inserts were 906bp in length and consisted of partial 18S,28Sand complete ITS1(353bp),5.8S(157bp)and ITS2(299bp)rDNA sequences.The similarity in ITS1and ITS2sequences among the Anisakid larvae sample and Pseudoterranova decipiens available in GenBank were over 99.7%,and lower homology with other nematodes.The results of the present study provided a foundation for further studies of Anisakid larvae.