A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa. Issue 4 (August 2016) - Boubaker, Ghalia
Abstract: Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small ( rrn S) and large ( rrn L) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus ( rrn S + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus . Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes. Highlights: A novel 12/16-PCR allows specific identification of species and genotypes within the genus Echinococcus ( 12S rDNA -PCR). The 12/16-PCR allow the basic identification of a large range of taeniid cestodes including different species of the genus Echinococcus, Taenia and a few others. The 12/16-PCR were highly sensitive and had a detection limit of 1 pg for Echinococcus DNA. Based on unique and geographically stable discriminatory SNPs, G1 and G3 of Echinococcus granulosus sensu stricto can be discriminated. Another 25 SNPs allowed differentiation within Echinococcus canadensis (G6/7/8/10).