Abstract
BackgroundEvaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked.MethodsThe ΔΔCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses.ResultsUsing carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa.ConclusionThe SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.
Highlights
Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys
The Standard Operating Procedures (SOPs) reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys
Real-time PCRs were first optimised for each gene by varying both primer and PCR reagent concentrations, To further test the validity of the assays, the copy number of the Pfmdr1 gene was compared between the laboratory clones P. falciparum 3D7 and P. falciparum Dd2
Summary
Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. In areas of high transmission, where large numbers of malaria cases are presently inevitable, there is a reliance on anti-malarial drugs to treat the disease. Parasite resistance, especially of Plasmodium falciparum, has been recorded to every antimalarial drug currently in use [1]. It has been shown that differential copy numbers and/or differential transcription of putative drug resistance gene candidates may influence responses to drugs in malaria (page number not for citation purposes). Gene annotation actin (housekeeping) multidrug resistance protein calcium-transporting ATPase (SERCA) histamine-releasing factor, putative glutathione peroxidase glutathione reductase glutathione S-transferase fe-superoxide dismutase trx peroxidase (2-Cys peroxiredoxin) thioredoxin peroxidase.
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