用cox1基因片段的PCR鉴别亚洲带绦虫和牛带绦虫

The Distinction of Taenia asiatica from Taenia saginata by PCR of Mitochondrial cox1 Gene Fragment

目的:用cox1基因片段的PCR技术对亚洲带绦虫和牛带绦虫的快速鉴别。方法:用亚洲带绦虫和牛带绦虫线粒体cox1基因片段的特异性引物以及带绦虫cox1基因片段的通用引物,对贵州省都匀地区和从江地区的带绦虫虫卵和节片DNA进行常规PCR扩增,扩增产物经琼脂糖凝胶电泳检测和核酸序列测定,并用NC-BI Blast对扩增产物的核酸序列与NCBI数据库中带绦虫的cox1基因进行比对。结果:5株都匀带绦虫DNA样品经亚洲带绦虫cox1基因片段的特异性引物PCR,均扩增出约260 bp的产物,PCR产物的核酸序列与NCBI数据库中亚洲带绦虫线粒体cox1基因片段的同源性为100%;2株从江带绦虫的DNA样品用亚洲带绦虫特异性引物和牛带绦虫特异性引物的PCR均未见扩增产物,而通用引物PCR则扩增出约1 000 bp的产物,核酸序列与NCBI数据库中牛带绦虫线粒体cox1基因片段的同源性为99%,在牛带绦虫特异性引物的结合位点存在一个碱基差异。结论:常规PCR扩增特异性的cox1基因片段可快速鉴定亚洲带绦虫;从江牛带绦虫株的cox1基因特异性片段部分存在变异,导致与特异性引物结合能力的差异,可采用cox1基因通用引物PCR结合测序进行鉴定。

Objective： To distinguish Taenia asiatica from Taenia saginata by coxl gene fragment sequence amplified with conventional polymerase chain reaction （PCR） method. Methods： The mitochondrial coxl gene fragments of Duyun isolates and Congjiang isolates of Taenia asiatica and Taenia saginata were amplified by conventional PCR using specific primers and general primers analysis. The fragments were confirmed by agarose electrophoresis and sequencing analysis. The sequences were compared with those in NCBI database with NCBI Blast. Results： A 260bp fragment was amplified from DNA specimens of 5 Duyun isolates of Taenia, while no same fragment was amplified from DNA specimens of Congjiang isolates when using the same primers. The sequence of the 260bp fragment had a 100% identity with that of T. asiatica mitochondrial cox 1 gene reported in NCBI database. With the general primers for cox 1 gene of Taenia, a 1000bp fragment was amplified from DNA of Congjiang isolates and the sequence showed 99% identity with that of T. saginata mitochondrial coxl gene reported in NCBI database. Only one base variation was at the specific primer binding loci. Conclusion： T. asiatica could be identified rapidly by conventional PCR of the T. asiatica specific mitochondrial cox 1 gene fragment. But because of variations, this method was not always effective for T. saginata. In these cases, sequencing after PCR with the general primers might be useful.