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

GHALIA BOUBAKER; NATALIA MACCHIAROLI; LAURA PRADA; MARCELA A. CUCHER; MARA C. ROSENZVIT; ISKENDER ZIADINOV; PETER DEPLAZES; URMAS SAARMA; HAMOUDA BABBA; BRUNO GOTTSTEIN; MARKUS SPILIOTIS

PLOS NEGLECTED TROPICAL DISEASES

PUBLIC LIBRARY SCIENCE

Lugar: San Francisco; Año: 2013

1935-2735

Echinococcus granulosus is characterized by a 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 spatiotemporal 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 one-vial 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 ìg 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 (<40%) of the mPCR was low. 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.