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:	Ghalia Boubaker, Natalia Macchiaroli, Laura Prada, Marcela A Cucher, Mara C Rosenzvit, Iskender Ziadinov, Peter Deplazes, Urmas Saarma, Hamouda Babba, Bruno Gottstein, Markus Spiliotis
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
Acceso en línea:	https://doaj.org/article/140181023b444a45acf161881c792116
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spelling	oai:doaj.org-article:140181023b444a45acf161881c7921162021-11-18T09:15:27ZA multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.1935-27271935-273510.1371/journal.pntd.0002017https://doaj.org/article/140181023b444a45acf161881c7921162013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23350011/pdf/?tool=EBIhttps://doaj.org/toc/1935-2727https://doaj.org/toc/1935-2735Echinococcus 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.Ghalia BoubakerNatalia MacchiaroliLaura PradaMarcela A CucherMara C RosenzvitIskender ZiadinovPeter DeplazesUrmas SaarmaHamouda BabbaBruno GottsteinMarkus SpiliotisPublic Library of Science (PLoS)articleArctic medicine. Tropical medicineRC955-962Public aspects of medicineRA1-1270ENPLoS Neglected Tropical Diseases, Vol 7, Iss 1, p e2017 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270
spellingShingle	Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Ghalia Boubaker Natalia Macchiaroli Laura Prada Marcela A Cucher Mara C Rosenzvit Iskender Ziadinov Peter Deplazes Urmas Saarma Hamouda Babba Bruno Gottstein Markus Spiliotis A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.
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.
format	article
author	Ghalia Boubaker Natalia Macchiaroli Laura Prada Marcela A Cucher Mara C Rosenzvit Iskender Ziadinov Peter Deplazes Urmas Saarma Hamouda Babba Bruno Gottstein Markus Spiliotis
author_facet	Ghalia Boubaker Natalia Macchiaroli Laura Prada Marcela A Cucher Mara C Rosenzvit Iskender Ziadinov Peter Deplazes Urmas Saarma Hamouda Babba Bruno Gottstein Markus Spiliotis
author_sort	Ghalia Boubaker
title	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_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_sort	multiplex pcr for the simultaneous detection and genotyping of the echinococcus granulosus complex.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/140181023b444a45acf161881c792116
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A multiplex PCR for the simultaneous detection and genotyping of the Echinococcus granulosus complex.

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