Designing and developing a high-resolution melting technique for accurate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real-time PCR and in silico genetic evaluation

Abstract

Discrimination, accurate identification, and reliable techniques are required for accurate identification of Leishmania parasites. High-resolution melting (HRM) is recognized as an authentic and exact method. The main objective of this research was optimizing HRM analysis for detecting and screening Leishmania major, Leishmania tropica and mix infections. Thirty-six DNA samples of Leishmania parasite were prepared and analyzed. Two gene regions of amino acid permease 3 (AAP3) and cytochrome oxidase II (COII) were targeted and six pairs of specific new primers were designed. Bioinformatics analysis was employed to predict DNA temperature resolution for each species and compared with in-vitro results. The genetic diversity of the selected gene regions was analyzed using PCR-sequencing method and DnaSP 5.10.01 software. They were submitted in GenBank (KU680818- KU680821 and KY041643- KY041649). The haplotype diversity for both AAP3 and COII genes was 96% and 87%, respectively. Tajima's D index was 0.65 for AAP3 and 0.36 for COII. CLC Genomics Workbench 11 software predictions were significant and close to these findings. The designed primers could be able to identify at least two Leishmania species. Temperature variations in HRM technique separated Iranian Leishmania parasites of L. major, L. tropica and mix infections. The target genes and our modified HRM method proved this technique could be useful in both clinical and experimental settings. Also, it can be effective for detecting Leishmania parasites in different hosts such as humans, reservoir hosts and vectors. Indeed, HRM can be used as a technique in Leishmania identification as well as for ecological and epidemiological research.