A novel PCR-RFLP assay for molecular characterization of Echinococcus granulosus sensu lato and closely related species in developing countries

Cystic echinococcosis, due to Echinococcus granulosus sensu lato (s. l.), currently affects three million people, especially in low-income countries and results in high livestock production loss. DNA-based methods demonstrated genetic variability of E. granulosus s. l., and five species were recognized to belong to the complex, including E. granulosus sensu stricto (s.s) (genotypes G1–G3), Echinococcus equinus (genotype G4), Echinococcus ortleppi (genotype G5), Echinococcus canadensis (genotypes G6–G10), and the lion strain Echinococcus felidis. The characterization of Echinococcus species responsible for human and animal echinococcosis is crucial to adapt the preventive measures against this parasitic disease. The sequencing approach is the gold standard for genotyping assays. Unfortunately, developing countries do not often have access to these techniques. Based on in silico RFLP tools, we described an accurate PCR-RFLP method for Echinococcus spp. characterization. The double digestion with the HaeIII and HinfI restriction enzymes of the PCR product from nad1 gene (1071 bp) led to a clear discrimination between E. granulosus s. l. and most closely related species (Echinococcus shiquicus and Echinococcus multilocularis). Molecular procedures and phylogenetic analysis confirmed the efficiency and the reproducibility of this simple and fast PCR-RFLP method. This technique is proved useful for fresh/unfixed and FF-PET tissues and enables large-scale molecular epidemiological screening in developing countries.