Accurate Identification of Leishmania Parasites in Sand Flies by Polymorphism Analysis of Cytochrome Oxidase Subunit 2 Gene Using Polymerase Chain Reaction and Quantitative PCR-High Resolution Melting Techniques in Iranian Border with Iraq.

Abstract

Firmly identification of Leishmania in Phlebotomus papatasi and understanding of natural transmission cycles of parasites in sand flies are important for treatment and local control. Modified and developed method of High Resolution Melting (HRM) as a preferable technique was employed to accurate identification of Leishmania in sand flies from Iranian border with Iraq, by targeting cytochrome oxidase II (COII) gene and designing suitable primers. PCR products cloned into pTG19-T vector, then purified plasmid concentration was measured at 260 and 280nm wavelength. The melting curve plots were generated and DNA sequences were analyzed using Sequencher 3.1.1, CLC Main Workbench 5.5, MEGA 6, DnaSP5.10.01 and MedCalc® version 13.3.3 soft wares. Among about 3000 collected sand flies, 89 female Ph. papatasi were identified and two with L. major. In amplified fragment of COII gene among 611bp, 452bp had no genetic variations with low polymorphic sites (P= 0.001) and high synonymous (79.8%) as compare to non-synonymous sites (20.2%). Leishmania major was discriminated in Ph. papatasi with 0.84 °C melting temperature (Tm) and unique curve based on thermodynamic differences was an important criterion using HRM technique. Subsequent war in Iraq made a high risk habitat for parasites transmission. It is important to discover accurate diagnostic procedures for leishmaniasis control.