Pfmdr1 and Pvmdr1 gene copy estimation

The pfmdr1 gene copy number of P. falciparum was assessed by real-time PCR. Genomic DNA of P. falciparum clones 3D7 (which has a single copy of pfmdr1) was used as a calibrator and pfβ-tubulin, a house-keeping gene, was used as an internal control. The primers for pfmdr1 and β-tubulin were described previously [65]. For P. vivax, the Salvador I strain was used as a calibrator and the pvaldolase gene, which is known to be a single copy gene in P. vivax, was used as an internal control using the published primers [66].

Amplification was performed in triplicate in a total volume of 20 μl containing 10μl of SYBR Green PCR Master Mix, 0.75 μl of each of the sense and anti-sense primers (10 μM), 20 ng of genomic DNA and 3.5 μl of water. Reaction was performed in CFX96 Touch^™ Real-Time PCR Detection System (Bio-Rad), with an initial denaturation at 95°C for 3 min, followed by 45 cycles at 94°C for 30 sec, 55°C for 30 sec, and 68°C for 1 min with a final 95°C for 10 sec. This was then followed by a melting curve step of temperature ranged from 65°C to 95°C with 0.5°C increment to determine the melting temperature of each amplified product. A negative control with no template was used in each run. Each sample was run in triplicates and the C_t values and melting temperature were recorded at the end of the reactions. The average and standard deviation of the three C_t values were calculated, and the average value was accepted if the standard deviation was lower than 0.32. The 2^ΔΔCt±SD method for relative quantification was used to estimate the gene copy number [66] and the results were expressed as the N-fold copy number of the targeted gene in relative to the reference. Fisher’s exact test (given small sample size) was used to test for significant differences in mutation prevalence and gene copy number among the study populations. All statistical analyses were performed in R (R Core Team 2013).