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A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex

Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canade...

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Autores principales: Boubaker, Ghalia, Macchiaroli, Natalia, Prada, Laura, Cucher, Marcela A., Rosenzvit, Mara C., Ziadinov, Iskender, Deplazes, Peter, Saarma, Urmas, Babba, Hamouda, Gottstein, Bruno, Spiliotis, Markus
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547860/
https://www.ncbi.nlm.nih.gov/pubmed/23350011
http://dx.doi.org/10.1371/journal.pntd.0002017
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author Boubaker, Ghalia
Macchiaroli, Natalia
Prada, Laura
Cucher, Marcela A.
Rosenzvit, Mara C.
Ziadinov, Iskender
Deplazes, Peter
Saarma, Urmas
Babba, Hamouda
Gottstein, Bruno
Spiliotis, Markus
author_facet Boubaker, Ghalia
Macchiaroli, Natalia
Prada, Laura
Cucher, Marcela A.
Rosenzvit, Mara C.
Ziadinov, Iskender
Deplazes, Peter
Saarma, Urmas
Babba, Hamouda
Gottstein, Bruno
Spiliotis, Markus
author_sort Boubaker, Ghalia
collection PubMed
description Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus.
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spelling pubmed-35478602013-01-24 A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex Boubaker, Ghalia Macchiaroli, Natalia Prada, Laura Cucher, Marcela A. Rosenzvit, Mara C. Ziadinov, Iskender Deplazes, Peter Saarma, Urmas Babba, Hamouda Gottstein, Bruno Spiliotis, Markus PLoS Negl Trop Dis Research Article Echinococcus granulosus is characterized by high intra-specific variability (genotypes G1–G10) and according to the new molecular phylogeny of the genus Echinococcus, the E. granulosus complex has been divided into E. granulosus sensu stricto (G1–G3), E. equinus (G4), E. ortleppi (G5), and E. canadensis (G6–G10). The molecular characterization of E. granulosus isolates is fundamental to understand the spatio-temporal epidemiology of this complex in many endemic areas with the simultaneous occurrence of different Echinococcus species and genotypes. To simplify the genotyping of the E. granulosus complex we developed a single-tube multiplex PCR (mPCR) allowing three levels of discrimination: (i) Echinococcus genus, (ii) E. granulosus complex in common, and (iii) the specific genotype within the E. granulosus complex. The methodology was established with known DNA samples of the different strains/genotypes, confirmed on 42 already genotyped samples (Spain: 22 and Bulgaria: 20) and then successfully applied on 153 unknown samples (Tunisia: 114, Algeria: 26 and Argentina: 13). The sensitivity threshold of the mPCR was found to be 5 ng Echinoccoccus DNA in a mixture of up to 1 µg of foreign DNA and the specificity was 100% when template DNA from closely related members of the genus Taenia was used. Additionally to DNA samples, the mPCR can be carried out directly on boiled hydatid fluid or on alkaline-lysed frozen or fixed protoscoleces, thus avoiding classical DNA extractions. However, when using Echinococcus eggs obtained from fecal samples of infected dogs, the sensitivity of the mPCR was low (<40%). Thus, except for copro analysis, the mPCR described here has a high potential for a worldwide application in large-scale molecular epidemiological studies on the Echinococcus genus. Public Library of Science 2013-01-17 /pmc/articles/PMC3547860/ /pubmed/23350011 http://dx.doi.org/10.1371/journal.pntd.0002017 Text en © 2013 Boubaker et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Boubaker, Ghalia
Macchiaroli, Natalia
Prada, Laura
Cucher, Marcela A.
Rosenzvit, Mara C.
Ziadinov, Iskender
Deplazes, Peter
Saarma, Urmas
Babba, Hamouda
Gottstein, Bruno
Spiliotis, Markus
A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
title A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
title_full A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
title_fullStr A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
title_full_unstemmed A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
title_short A Multiplex PCR for the Simultaneous Detection and Genotyping of the Echinococcus granulosus Complex
title_sort multiplex pcr for the simultaneous detection and genotyping of the echinococcus granulosus complex
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547860/
https://www.ncbi.nlm.nih.gov/pubmed/23350011
http://dx.doi.org/10.1371/journal.pntd.0002017
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