Simple and Direct Characterization of Leishmania donovani Isolates Based on Cytochrome Oxidase II Gene Sequences

We primarily identified Leishmania donovani parasites from eastern Sudan using species-specific primers that amplify a whole length minicircle. Based on the amplification of a cytochrome oxidase II fragment (COII), heteroduplex analysis (HDA) was performed. In HDA, the appearance of the extra bands with molecular weights higher than 540 bp indicates the presence of mismatched bases in the selected samples. Such bands were detected when hybridization was between reference strains and clinical isolates, as well as between the reference strains themselves, while no heteroduplexes were detected between the clinical isolates. Moreover, an RFLP assay using the restriction enzyme Ssp1 was performed on the original 540 bp products to discern an A-G transition, which differentiates between members of the Leishmania (L) infantum and those of Leishmania (L) donovani subspecies. The proposed minicircle genes-based analysis was rapid and easy to perform method for the characterization of Leishmania donovani complex isolates and with a potential to be extended to characterization of other species of Leishmania.