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

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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.