Identification of chloroquine resistance Pfcrt-K76T and determination of Pfmdr1-N86Y copy number by SYBR Green I qPCR

Objective: To identify prevalence of chloroquine resistance point mutation at (Pfcrt, K76T) and (Pfmdr1, N86Y) copy number variation. Methods: SYBR Green I based real time PCR was used. One hundred and thirty-three samples were analyzed for (Pfcrt, K76T) and (Pfmdr1, N86Y) copy number from dried blood spot. Parasite DNA was extracted using high pure DNA preparation kit. The amplification of DNA was done by using AccuPower 2× GreenStar™ qPCR Master mix. For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI. Absolute quantification method was used to determine the Pfmdr1-N86Y copy number variations. Standard curve was built from strain 3D7 gDNA since it has single copy of Pfmdr1 per haploid genome. The known positive controls with single and multi-copy number of Pfmdr1 gene were included in each experiment. The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation. Results: Out of 133 samples 73 (54.89%) were confirmed as mutant (Pfcrt, 76T) and the remaining 60 (45.11%) were genotyped as wild type (Pfcrt, K76). The (Pfmdr1, N86Y) copy number variation was determined for 133 clinical samples. Out of these samples 61 (45.86%) had single copy and the remaining 72 (54.14%) had multi-copy numbers higher than 1.5 copies per genome. Thirty-four (25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38 (28.57%) were more than 2.5 copies per genome. The minimum and maximum copies per genome were 0.474 and 4.741, respectively. Conclusions: The study showed high prevalence level and fixation of Pfcrt, 76T mutation after chloroquine withdrawal. The prevalence of Pfmdr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers. However, the prevalence level was not statistically significant.