广西融水县并殖吸虫虫种的分子鉴别

Molecular identification of Paragonimus species from Rongshui county Guanqli Province

用分子生物学方法鉴定广西融水县大小两种并殖吸虫囊蚴感染动物后所获成虫的虫种。在广西融水县采集溪蟹分离获得大小两种囊蚴 ,感染大鼠后获成虫 ,曾从形态上鉴定为斯氏狸殖吸虫和巨睾并殖吸虫。但用酚 -氯仿法抽提成虫基因组DNA ,PCR扩增第二内转录间隔区 (ITS2 )基因后测序 ,用Blast程序与GenBank中的斯氏狸殖吸虫、巨睾并殖吸虫等的ITS2基因序列进行同源性检索 ,用PrimerPremier软件比较其基因差异 ,用Phylip软件的Neighbor程序绘制其基因进化树后 ,测得大小囊蚴感染所获成虫的ITS2基因片段分别为 4 37bp、4 33bp ,两者的基因同源性为 10 0 % ,与斯氏狸殖吸虫的基因同源性分别为 10 0 %、99% ,与巨睾并殖吸虫的基因同源性分别为 91%、92 % ;基因序列差异比较 ,大小囊蚴感染所获成虫与斯氏狸殖吸虫的基因序列差异为 0 ,而与巨睾并殖吸虫存在明显差异 ;绘制基因进化树 ,大小囊蚴感染所获成虫与斯氏狸殖吸虫在同一分支同一位置 ,与巨睾并殖吸虫亲缘关系较远。融水县大小囊蚴感染所获成虫与斯氏狸殖吸虫为同一物种 ,小囊蚴感染所获成虫并非巨睾并殖吸虫。

By using the molecular biology techniques,the species of worms collected from animals infected by the big and small cysts in Guangxi province were identified.These big or small cysts were isolated from freshwater crabs in Rongshui district,and then rats were infected with these cysts to obtain adult worms,that were identified by morphology as Paragonimus shrjabini and Paragonimus macrorchis.After the DNA of the adult worms was extracted by phenol-choloroform method,the nuclear ribosomal DNA was amplified in the second internal transcribed spacer (ITS2) by PCR,and then was sequenced.The gene homologies were compared with those of the Blast program of GenBank,and the gene variations were compared with those of the Primer-Prermer software.Thus,the phylogenetic tree were set up with Phylip software.It was found that the ITS2 lengths of the adult worms infected with big or small cysts were 437 bp and 433 bp respectively.The gene homology of the adult worms infected with big and small cysts was 100%;those with P. skrjabini were 100% and 99% respectively; those with P.macrorchis were 91% and 92% respectively.The comparison of gene variations indicated that the gene variation number was 0 among P.skrjabini and them,but the difference was obvious in comparison with P.macrorchis.The phylogenetic trees showed that adult worms infected with big and small cysts and P skrjabini located at the same branch location,but quite far from the phylogenetic location of P.macrorchis.It concludes that P.skrjabini and the worms infected with big or small cysts were the same species,and the worms infected by small cysts are not P.macrorchis.