Differentiation of three species of Leishmania major, L. tropica and L. infantum by PCR-RFLP technique

Background: The Leishmaniasis is a zoonosis disease with a global spread that occurs in two, cutaneous and visceral forms. In Iran, three species of Leishmania tropica, Leishmania major, and Leishmania infantum has been reported from different regions of the country. Nowadays, molecular methods are usually used for the diagnosis and identification of parasite species. Amplification of the ITS۱ gene can also be used to differentiate symptomatic and asymptomatic visceral leishmaniasis as well as types of cutaneous leishmaniasis. Objectives: The main aim of our research was to diagnose and identify the common Leishmania species in Iran through ITS۱ gene amplification and using the PCR-RFLP method. Methods: First, L. major, L. tropica, and L. infantum parasites were proliferated in an RPMI culture medium, and DNA was extracted from these species separately then ITS۱ gene of the parasite was amplified using the PCR-RFLP method. Results: The result of PCR-RFLP after enzymatic cutting indicated, L. tropica produced ۴ fragments of ۱۳۹, ۷۶, ۵۶, ۲۰ bp bands; L. major showed two fragments of ۱۶۵, ۱۳۹ bp bands and L. infantum produced three fragments of ۱۴۱, ۹۱, ۵۴ bp bands. Conclusion: The results of the present study showed that the use of ITS۱ gene and HaeIII enzyme in the PCR-RFLP method is efficient for identifying L. tropica, L. major, and L. infantum.

Keywords:

Leishmania major , L. tropica , L. infantum , RFLP-PCR , ITS۱-gene