【摘要】 提取我国广西、四川、黑龙江、甘肃等地及法国的片形吸虫的总 DNA,用 PCR扩增完整的 ITS- 2。对得到的 PCR产物用限制性内切酶 Hsp92 ,Rca 进行酶切和 RFL P技术分析。并对 ITS- 2 PCR产物进行双向测序。以便确定国内片形吸虫种内及种间 ITS- 2的特点和序列变异的水平。结果显示 :广西、四川、黑龙江、法国等地的样品通过 PCR均扩增到约 5 5 0 bp的 ITS- 2目标片段。Hsp92 ,Rcal可区别不同种的片形吸虫。对PCR产物的序列分析结果表明片形吸虫 ITS- 2全长均为 36 2 bp,同种内 ITS- 2序列无变异 ,不同种间 ITS- 2序列存在 5～ 6个碱基差异 ,变异率约为 2 .8%。对各地区片形吸虫 ITS- 2的 PCR— RFL P分析与对 PCR产物的 DNA序列分析一致证明 ,来自四川的样品和法国样品同属肝片形吸虫 F.hepatica;广西样品属大片形吸虫 F.gigantica;黑龙江的样品为中间型片形吸虫。本研究首次从分子水平上证实我国除有肝片形吸虫、大片形吸虫外 ,还存在中间型的片形吸虫更多还原

【Abstract】 In present study, the total DNA of Fasciola spp isolated from Guangxi, Sichuan, Heilongjiang, Gansu of China and France was isolated by CTAB method. A pair of primers was designed according to the published sequence of ITS2 of ribosomal RNA gene. Restriction enzyme Hsp92Ⅱ and RcaⅠ were used to digest the PCR products. The PCR products were cloned in the vector of pGEM Teasy by standard method. The recombinant clones were sequenced double strandedly on ABI 377 DNA sequencer. The results showed All Fasciola isolates gave a PCR product of about 550 bp. The PCR products were digested by Hsp92Ⅱ and Rcal, respectively. As a result, all Fasciola spp studied in this work could be classified into three guoups. Sequencing analysis of all the PCR products further confirmed same classification. The ITS2 of all Fasciola spp was 362 bp in length. Sequence comparison also revealed that there were no variations in ITS2 sequence within one species of Fasciola and there were five to six bases difference in ITS2 sequence between different species of Fasciola. It was concluded that the Fasciola istolates from Sichuan and France were F. hepatica , the Guangxi Fasciola isolates were F. gigantica and the Heilongjiang Fasciola isolates were intermediate Fasciola. This is the first time to confirm that there are intermediate Fasciola in China in addition of F. hepatica and F. gigantica .更多还原