Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene

Emi Okamoto, Michiyo Tashiro, Pedro Ortiz, Uday Kumar Mohanta, Cristian Hobán, César A. Murga-Moreno, José M. Angulo-Tisoc, Madoka Ichikawa-Seki

Research output: Contribution to journalArticlepeer-review

Abstract

Background: Multiplex polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) for nuclear phosphoenolpyruvate carboxykinase (pepck) and polymerase delta (pold), respectively, have been used to differentiate Fasciola hepatica, F. gigantica, and hybrid Fasciola flukes. However, discrimination errors have been reported in both methods. This study aimed to develop a multiplex PCR based on a novel nuclear marker, the fatty acid binding protein type I (FABP) type I gene. Methods: Nucleotide sequence variations of FABP type I were analyzed using DNA samples of F. hepatica, F. gigantica, and hybrid Fasciola flukes obtained from 11 countries in Europe, Latin America, Africa, and Asia. A common forward primer for F. hepatica and F. gigantica and two specific reverse primers for F. hepatica and F. gigantica were designed for multiplex PCR. Results: Specific fragments of F. hepatica (290 bp) and F. gigantica (190 bp) were successfully amplified using multiplex PCR. However, the hybrid flukes contained fragments of both species. The multiplex PCR for FABP type I could precisely discriminate the 1312 Fasciola samples used in this study. Notably, no discrimination errors were observed with this novel method. Conclusions: Multiplex PCR for FABP type I can be used as a species discrimination marker in place of pepck and pold. The robustness of the species-specific primer should be continuously examined using a larger number of Fasciola flukes worldwide in the future since nucleotide substitutions in the primer regions may cause amplification errors. Graphical abstract: [Figure not available: see fulltext.].

Original languageEnglish
Article number379
JournalParasites and Vectors
Volume15
Issue number1
DOIs
StatePublished - Dec 2022
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by a Grant-in-Aid for Scientific Research (C) from MEXT KAKENHI (grant number 21K05954) and the Tohoku Initiative for Fostering Global Researchers for Interdisciplinary Sciences (TI FRIS) of MEXT's Strategic Professional Development Program for Young Researchers.

Publisher Copyright:
© 2022, The Author(s).

Keywords

  • FABP type I
  • Fasciola
  • Genotyping
  • Multiplex PCR

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