利用PCR-RFLP技术鉴别异尖线虫

应用PCR-RFLP技术对福建沿海海鱼中常见的简单异尖线虫、典型异尖线虫、对盲囊线虫和针蛔线虫幼虫进行鉴定.采用通用引物扩增上述4种异尖线虫幼虫的核糖体DNA内转录间隔区(ITS)序列,对PCR产物进行回收、克隆和测序.根据序列分析结果,选用限制性内切酶HinfⅠ和RsaⅠ进行酶切,酶切产物经琼脂糖电泳分析.结果表明:同时使用HinfⅠ和RsaⅠ酶,可以将4种异尖线虫进行分类;应用特异性引物扩增这4种异尖线虫,获得了预期扩增片段.可见,根据4种线虫ITS序列建立的PCR-RFLP技术能够对这4种异尖线虫进行准确、特异和快速的鉴定.

Genetic identification of Anisakidae nematodes by PCR-RFLP

Anisakis simplex,A.typica,Contracaccum sp.and Raphidascaris trichiuri,which were commonly found in Fujian coastal marine fish,were identified by polymerase chain reaction linked restriction fragment length polymorphism(PCR-RFLP).Using a pair of conserved primers,the sequences of the internal transcribed spacer(ITS) of Anisakidae nematodes were amplified by PCR.The amplicons were purified,reclaimed,cloned,and digested by HinfⅠ and RsaⅠ selected according to the analysis of ITS sequences,followed by agarose gel electrophoresis analysis.It showed that the four nematodes could be classified by using HinfⅠ and RsaⅠ.The expected fragments of these nematodes were amplified by specific primers.It had been proved that the PCR-RFLP technique based on the ITS gene sequences was a simple,specific method for the identification of these nematodes.