Sequencing and Phylogenetic analysis of ITS-rDNA gene of Leishmania parasites in human in north of Khuzestan

Introduction & Objective: Leishmania parasites, agents of Cutaneous Leishmaniasis (CL) in humanin north of Khuzestan were identified and typed molecularly by targeting ITS-rDNA gene.Materials and Methods: Samples were collected from suspected people to CL in different villagesand cities of North of Khuzestan. Stained Smears were examined under a light microscope and classified based on frequency of Leishmania amastigotes. DNA of parasite extracted and ITS-rDNA gene amplified by nested PCR. The PCR product digested with BsuRI restriction enzyme which was selected based on in-silico analysis using CLC DNA workbench software. To confirm the results of PCR-RFLP, some PCR products were sequenced in both directions using the ITS1F and ITS2R4 primers directly. Individual contig sequences were aligned with GenBank sequences of all regional species using Sequencher 4.4 software for PC (Gene Codes Corp, Ann Arbor, Michigan, USA), and unique haplotype were identified after export into MEGA software for phylogenetic analysis. Results and Conclusions: All microscopically-positive and some negative samples were examined to amplify ITS-rDNA gene. Using RFLP and sequencing L. major was firmly identified and some others which are not L. major, will be identified after sequencing. The results of reliable molecular techniques and phylogenetic analyses used in this project could determine the advantages of molecular tools to conventional methods to typing Leishmania parasites in human.

کلیدواژه ها:

Leishmania major ، ITS-rDNA ، PCR-RFLP ، Phylogenetic analysis and Khuzestan