Molecular identification and phylogenetic analysis of nuclear rDNA sequences among three opisthorchid liver fluke species (Opisthorchiidae: Trematoda)

Seokha Kang, Tahera Sultana, Valery B. Loktev, Surasakdi Wongratanacheewin, Woon Mok Sohn, Keeseon S. Eom, Joong Ki Park

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53 Scopus citations


In this study, we describe the development of a fast and accurate molecular identification system for human-associated liver fluke species (Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) using the PCR-RFLP analysis of the 18S-ITS1-5.8S nuclear ribosomal DNA region. Based on sequence variation in the target rDNA region, we selected three species-specific restriction enzymes within the ITS1 regions, generating different restriction profiles among the species: MunI for O. viverrini, NheI for O. felineus, and XhoI for C. sinensis, respectively. Each restriction enzyme generated different-sized fragments specific to the species examined, but no intraspecific polymorphism or cross-reaction between the species was detected in their restriction pattern. These results indicate that PCR-linked restriction analysis of the ITS1 region allows for the rapid and reliable molecular identification among these opisthorchid taxa. In addition, phylogenetic analysis of rDNA sequences using different methods (MP, ML, NJ, and Bayesian inference) displayed O. viverrini and O. felineus as a sister group, but this relationship was not strongly supported. The failure of recovering a robust phylogeny may be due to the relatively small number of synapomorphic characters shared among the species, yielding weak phylogenetic signal. Alternatively, rapid speciation within a very short period time could be another explanation for the relatively poorly resolved relationships among these species. Our data are insufficient for discriminating between sudden cladogenesis and other potential causes of poor resolution. Further information from independent loci might help resolve this phylogeny.

Original languageEnglish
Pages (from-to)191-197
Number of pages7
JournalParasitology International
Issue number2
StatePublished - Jun 2008

Bibliographical note

Funding Information:
Constructive comments from the editor and two anonymous reviewers helped improve the manuscript. We thank Steve Nadler for his careful reading of the manuscript. DNA samples used in the present study were deposited in the Parasite Resource Bank of Korea National Research Resource Center, Republic of Korea. This work was supported by a Korea Research Foundation Grant (KRF-2005-070-C00124) and the research grant of the Chungbuk National University in 2007 to JKP.


  • Clonorchis
  • ITS1
  • Molecular identification
  • Molecular phylogeny
  • Opisthorchis
  • Trematoda


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