Eight microsatellite loci markers were preformed in the study. Semi-nested PCR was used to amplify P. vivax with seven markers: Pv1.501, Pv3.27, Pv6.34, Pv 8.504, Pv14.297, Pv3.502, Pv11.162 and single PCR was used for MS1. In a total volume of 20 ul with 2 ul of genomic DNA template, 2U of Platinum Taq polymerase,1X buffer, 2.5 mM MgCl2, 250 uM of each oligonucleotide primers, 125uM dNTP, and then 1 ul of product from first-round amplification was used to initiate the second round amplification. Cycling parameter for PCR was as follows: 95 ?C for 5 min, 94 ?C 30 s, 52?C 30 s, 72 ?C 30 s, then 72 ?C for 2 min. 25 cycles are used in both PCR. For single PCR, 2 ?l of genomic DNA template, 2 U of Platinum Taq polymerase, 2mM MgCl2, 1× reaction buffer and 0.1 mM of each dNTP. Cycling parameters were the same for all primer pairs: 1 cycle at 94 °C for 2 min, 40 cycles at 94 °C for 30 s, 58 °C for 40 s and 72 °C for 30 s, and a final cycle at 72 °C for 5 min.
ABI 3130 Genetic analyzer and GeneMapper® software version 4.0 (Applied Biosystems) was used to measure an allele in each locus where is the products were sized by comparison to LIZ-500 size standards. Data analysis is using the predominant alleles presented in each locus. If an isolate had more than one allele at any a locus, the minor allele was respected as a signal of multiple infections in case of height was at least one-third of the predominant allele 13. Multiplicity of infection (MOI) of a given isolate was measured as the highest number of observed alleles at any of the 8 loci. The mean number of alleles calculated from the only of predominant alleles at each locus. The expected heterozygosity (HE), the formula defined as He = 1/(1-n) (1-?pi2) where p is the frequency of ith allele. Expected heterozygosity (He) ranges between 0 and 1, a value close to 1 indicated high genetic diversity levels in the population 14, were calculated using FSTAT v.2.9.3 15. For number of alleles per locus values were calculated from all detected alleles at each loci and then divided by the total number of samples. Multilocus linkage disequilibrium (LD) was calculated by using a standardized index of association (ISA) 16, 17. Only the dominant alleles were considered to verify linkage. This test compares the variance (VD) of the number of alleles shared between all pairs of haplotypes observed in the population (D) with the variance expected under random association of alleles (VE) as follows: ISA = (VD/VE- 1) (r-1), where r is the number of loci analyzed This analysis was performed using the LIAN 3.7 software 18.