Specie identification from single amphistome eggs by PCR-RFLP based on ribosomal DNA ITS-2 marker

基于核糖体 DNA ITS-2 标记的 PCR-RFLP 技术对单个双足虫卵进行物种鉴定

• 批准号: 10660278
• 负责人: ITAGAKI Tadashi
• 金额: $ 1.79万
• 依托单位: IWATE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660278/
• 关键词: amphistome; Calicophoron calicophorum; Orthocoelium streptocoelium; Homalogaster paloniae; Ribosomal DNA; Nucleotide sequence; ITS-2; PCR-RFLP; Orthocoelium streptocoelium; Homalogaster paloniae; Calicophoron microbothlium; DNA

The purpose of this study is first to describe the extent of ITS2 sequence difference among 3 species of amphistomes, namely Calicophoron calicophorum, Orthocoelium streptocoelium and Homalogaster paloniae, and secondly to assess PCR-RFLP method for the species identification of single eggs of the parasites. The length of ITS2 sequence was 284bp in C. calicophoronm and O. streptocoelium, and 285bp in H. paloniae. The ITS2 sequences of the three species contained 19variable sites including one gap and sequence difference between species was 4.2-5.3%. Restriction maps were constructed for ITS2 sequences of the three species. Although several sites existed for a number of common restriction endonucleases, the three species of amphistomes could be distinguished from each other by the position of Acc II, Mfl I and Hha I. These results suggest that ITS2 sequence should be useful as DNA marker for species identification of single eggs of the three species using PCR-RFLP technique.

本研究的目的首先是描述 3 种双栖动物（即 Calicophoron calicophorum、Orthocoelium streptocoelium 和 Homalogaster paloniae）之间 ITS2 序列差异的程度，其次评估 PCR-RFLP 方法对寄生虫单卵的物种鉴定。 ITS2序列的长度在C. calicophoronm和O. streptocoelium中为284bp，在H. paloniae中为285bp。 3个物种的ITS2序列包含19个可变位点，其中1个缺口，物种间序列差异为4.2-5.3%。为这三个物种的 ITS2 序列构建了限制性图谱。尽管存在一些常见限制性内切酶的多个位点，但这三种双栖动物可以通过 Acc II、Mfl I 和 Hha I 的位置来区分。这些结果表明，ITS2 序列应可作为 DNA 标记，用于使用 PCR-RFLP 技术对这三个物种的单个卵进行物种鉴定。

项目成果

Itagaki, T., N. Tsumagari , K.Tsutsumi: "Discrimination of three amphistome species by PCR-RFLP based on rDNA ITS-2 marker Discrimination of three amphistome species by PCR-RFLP based on rDNA ITS-2 marker"submitteed to Parasitology Research.

Itagaki, T.、N. Tsumagari、K.Tsutsumi：“基于 rDNA ITS-2 标记通过 PCR-RFLP 区分三种两栖动物物种 基于 rDNA ITS-2 标记通过 PCR-RFLP 区分三种两栖动物物种”提交给 Parasitology

Itagaki, T., Tsumagari, Y. and Tsutsumi, K: "Species differentiation of paramphistomoidea based on PCR-linked RFLP of ITS-2 of ribosomal DNA"Parasitology International. 47 (suppl). (1998)

Itagaki, T.、Tsumagari, Y. 和 Tsutsumi, K：“基于核糖体 DNA ITS-2 的 PCR 连接 RFLP 的副蚜总科物种分化”国际寄生虫学。