Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum.
BACKGROUND
Evaluating 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.


METHODS
The DeltaDeltaCt 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.


RESULTS
Using 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.


CONCLUSIONS
The 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.
Ferreira
Isabel D
ID
Centro de Malária e Outras Doenças Tropicais/IHMT/UNL, Rua da Junqueira, 96, 1349-008, Lisbon, Portugal. ferreiraID@ihmt.unl.pt
Rosário
Virgílio E do
VE
Cravo
Pedro V L
PV
D000818:Animals; D052160:Benzothiazoles; D014408:Biomarkers, Tumor / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000097:blood; D003959:Diamines; D004351:Drug Resistance / Q000235:genetics*; D005456:Fluorescent Dyes / Q000191:economics; D018628:Gene Dosage / Q000187:drug effects / Q000235:genetics*; D019450:Genes, MDR / Q000235:genetics; D005980:Glutathione Reductase / Q000235:genetics; D006801:Humans; D009930:Organic Chemicals / Q000191:economics; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011804:Quinolines; D015203:Reproducibility of Results; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics; D012680:Sensitivity and Specificity; D000090324:Tumor Protein, Translationally-Controlled 1
D052160:Benzothiazoles; D014408:Biomarkers, Tumor; D017931:DNA Primers; D016054:DNA, Protozoan; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D000090324:Tumor Protein, Translationally-Controlled 1; C098022:SYBR Green I; D005980:Glutathione Reductase; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases