Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene
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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2022-10-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13071-022-05538-7 |
| _version_ | 1797985977412616192 |
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| author | Emi Okamoto Michiyo Tashiro Pedro Ortiz Uday Kumar Mohanta Cristian Hobán César A. Murga-Moreno José M. Angulo-Tisoc Madoka Ichikawa-Seki |
| author_facet | Emi Okamoto Michiyo Tashiro Pedro Ortiz Uday Kumar Mohanta Cristian Hobán César A. Murga-Moreno José M. Angulo-Tisoc Madoka Ichikawa-Seki |
| author_sort | Emi Okamoto |
| collection | DOAJ |
| description | 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 |
| first_indexed | 2024-04-11T07:26:38Z |
| format | Article |
| id | doaj.art-969fb7f63e3841b1a2c067aaa3a441b8 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-04-11T07:26:38Z |
| publishDate | 2022-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-969fb7f63e3841b1a2c067aaa3a441b82022-12-22T04:37:04ZengBMCParasites & Vectors1756-33052022-10-011511810.1186/s13071-022-05538-7Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I geneEmi Okamoto0Michiyo Tashiro1Pedro Ortiz2Uday Kumar Mohanta3Cristian Hobán4César A. Murga-Moreno5José M. Angulo-Tisoc6Madoka Ichikawa-Seki7Iwate UniversityIwate UniversityUniversidad Nacional de CajamarcaSher-E-Bangla Agricultural UniversityUniversidad Nacional de CajamarcaUniversidad Nacional de CajamarcaUniversidad Nacional Mayor de San MarcosIwate UniversityAbstract 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 abstracthttps://doi.org/10.1186/s13071-022-05538-7FasciolaMultiplex PCRGenotypingFABP type I |
| spellingShingle | Emi Okamoto Michiyo Tashiro Pedro Ortiz Uday Kumar Mohanta Cristian Hobán César A. Murga-Moreno José M. Angulo-Tisoc Madoka Ichikawa-Seki Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene Parasites & Vectors Fasciola Multiplex PCR Genotyping FABP type I |
| title | Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene |
| title_full | Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene |
| title_fullStr | Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene |
| title_full_unstemmed | Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene |
| title_short | Development of novel DNA marker for species discrimination of Fasciola flukes based on the fatty acid binding protein type I gene |
| title_sort | development of novel dna marker for species discrimination of fasciola flukes based on the fatty acid binding protein type i gene |
| topic | Fasciola Multiplex PCR Genotyping FABP type I |
| url | https://doi.org/10.1186/s13071-022-05538-7 |
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