Allelic Variations of Plasmodium vivax Apical Membrane Antigen-1 (Pv AMA-1) in Malarious Areas of Southeastern Iran Using PCR-RFLP Technique

Abstract

Background: Although Plasmodium vivax is usually known as benign malaria, some variations of the parasite can result in acute and sever infection. In this study we tried to determine some genetic variations in PvAMA-1 antigen among the samples were collected form southeastern Iran.

Methods: About two ml blood samples were collected into EDTA pre-dosed tubes from 30 P. vivax-infected patients individually between 2011 and 2013. A Giemsa stained thick and thin blood film was prepared from each of the patients. A PCR-RFLP technique was employed using EcoR-1, Pvu-II and Hind3 restriction enzymes to determine the allelic variations of the antigen.

Results: A 1300bp gene corresponding to PvAMA-1 was selected for the amplification process. Among the total cases identified in this study 90% showed similar bounds when exposed to the restriction enzymes. Nine isolates (accession numbers: KF435081-KF435083 and JF682785-JF682790) were identified and registered in Gene bank. Identity among isolates was more than 96% in nucleotide level. Dendrogram clarified a close relationship among the clusters in spite of geographical distribution of the parasite.

Conclusion: This study increased our data about prevalence and variation of PvAMA-1 alleles amongst P. vivax isolates in southeastern parts of Iran where besides native population bears considerable Afghan and Pakistani immigrants.

Keywords: Allelic variations; Iran; PCR-RFLP; PvAMA-1.

Fig. 1:

Electrophoretic product of PvAMA-1 gene using PCR technique including columns 1&2 amplified PvAMA-1 gene with 1300bp and DNA size marker with 1000bp in Column 3

Fig. 2:

Electrophoretic product of PvAMA-1 gene treated with EcoR I, PvuII, and HindIII, enzymes using RFLP-PCR technique including Columns 1,2&3 treated with EcoRI. Column 4 main bound of PvAMA-1. Column 5 size marker with 1000bp. Columns 6, 7 and 8 treated with PvuII. Columns 9&10 treated with HindIII

Fig. 3:

The degree of identity for P.vivax AMA-1 gene among isolates from Iran (accession numbers: KF435081-3 and JF682785-90), Sal 1(XM_001615397) from Us, S3 (EF025195.1) from India and SK0814 (GU476488.1) from Korea in comparison with P. falciparum 3D7 AMA-1 gene (XM_001347979.1) as an outgroup

Fig. 4:

Dendrogram of P. vivax isolates taken from the present study and the other isolates from Us (XM_001615397), India (EF025195.1) and Korea (GU476488.1) in comparison with P. falciparum 3D7 AMA-1 gene (XM_001347979.1) as an outgroup based on sequence alignments of the mentioned samples.