application of high-concentration agarose gel electrophoresis on its-1 amplicons as a simple method for differentiation of species of the leishmania in iran

Leishmaniasis, a protozoan parasite that infects dogs, cats, and some rodents, is prevalent in many countries. diagnostic methods such as pcr, rflp, and real-time pcr have been tested on different leishmania species using different genes, particularly the its region of ribosomal dna. however, the weight of pcr products can differ between species. this study aimed to find a cost-efficient and easy method to separate smaller dna fragments on a higher percentage agarose gel compared to sequencing results.the dna was extracted using a commercial genomic dna extraction kit, followed by pcr reactions targeting the its-1 ribosomal dna gene of the leishmania. primers f (lsgits-1-f1: 5’-cattttccgatgattacac-3’) and r (lsgits-1r1: 5 -cgttatgtgagccgttatc-3) were used. the pcr-rflp method was used to identify leishmania species by digesting amplicon products using a restriction enzyme. samples were incubated for 14 hours at 37°c for enzymatic digestion. the digested products were examined under uv illumination on a 3% agarose gel for species identification.the expected bands of 266, 241, and 252 base pairs corresponding to the three common iranian species, including leishmania major, l. infantum, and l. tropica, respectively, were clearly separated and differentiated on the agarose gel (3%) in the presence of a 50 bp ladder. the results showed that the high-concentration agarose gel electrophoresis on its-1 amplicons can be used as a less expensive molecular method for differentiation of species of the leishmania spp. existing in iran compared to rflp pcr and sanger sequencing methods.