Algorithms based on PCR-RFLP of nad1 gene for genotyping of Echinococcus granulosus from human and animal isolates in Egypt

Background: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a simple rapid method for genotyping of Echinococcus garnulosus sensu lato (E. granulosus s. l.) in developing countries. Construction of algorithms based on PCR-RFLP using two restriction enzymes would be useful to study the genetic diversity of the parasite and would help in differentiation between ambiguous genotypes. Objective: The goal of the present work was to develop algorithms based on RFLP of nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 1 (nad1) sequences of reference genotypes of E. granulosus retrieved from GenBank for genotyping of human and animal isolates of E. granulosus in Egypt. Subjects and Methods: Retrieved nad1 sequences of reference genotypes were digested in silico individually with two restriction enzymes; Haemophilus influenza (HinfI) and Haemophilus aegyptius (HaeIII). The constructed PCR-RFLP algorithms were used for genotyping of 50 human and animal isolates (19 human, 23 camels and 8 pigs) analyzed by PCR-RFLP. To confirm the validity PCR-RFLP algorithms, samples corresponding to determined and undetermined genotypes as inferred from the algorithms were sequenced. Results: Utilizing PCR-RFLP and sequencing revealed that except for two cases (12.5%) which were typed as G1 among humans and one case as G5 in pigs (12.5%), G6 was the commonest genotype among human, camel and pig isolates collected. Conclusion: The algorithms based on PCR-RFLP of nad1 are valuable tools for genotyping ofE. granulosus s. l. especially with HinfI RFLP algorithm. Sequencing is still needed to reveal the genotypes of undetermined or ambiguous isolates.