DNA was extracted from 200 μL whole blood (venous blood collected in EDTA anti-coagulant) using QIAamp 96 DNA Blood Mini Kit (QIAGEN, Valencia, CA), and eluted in a final volume of 200 μL dH2O according to the supplier's instructions. nPCR was carried out as described [22 (link)].
PCR_LDR_FMA was carried out as described elsewhere [19 (link)]. Mixes of serially diluted plasmids containing inserts of P. falciparum, P. vivax, P. malariae or P. ovale 18 S rDNA were used as positive controls in addition to P. falciparum or P. vivax positive samples obtained from field isolates. The threshold for positivity for each species was determined using the mean value obtained from negative controls for each species, plus three times the standard deviation.
The primers and probes of the qPCR assay are listed in Table1 . In the design of a duplex qPCR, the probes combined in one reaction carried different fluorescent labels at their 5' ends. All four probes carried a black hole quencher (BHQ) at their 3'ends. The analytical specificity of primers and probes were evaluated for each Plasmodium species in silico by Blast searches and experimentally by using gDNA of the three alternatives Plasmodium species or of related blood borne parasites. To minimize costs of consumables, duplex reactions were performed in a final volume of 12.5 μL. Amplification and detection of the amplified product was performed in an iQcycling BioRad system, using iQSupermix from BioRad. The P. falciparum/P. vivax (Pf/Pv) duplex reaction contained 2.5 μL DNA (corresponding to 2.5 μL whole blood), 6.25 μL SuperMix iQ (BioRad), 0.35 μL Pf primer mix (50 μM), 0.35 μL of Pv primer mix 50 μM, 0.375 μL of Pf probe (10 μM), 0.375 μL of Pv probe (10 μM) and 2.3 μL double distilled water. The P. malariae/P. ovale (Pm/Po) duplex reaction contained equivalent amounts and concentrations of the respective primers and probes. The thermal profile used was 2 minutes at 50°C, followed by 10 minutes at 95°C and 45 cycles of 15 seconds at 95°C and 1 minute at 58°C.
PCR_LDR_FMA was carried out as described elsewhere [19 (link)]. Mixes of serially diluted plasmids containing inserts of P. falciparum, P. vivax, P. malariae or P. ovale 18 S rDNA were used as positive controls in addition to P. falciparum or P. vivax positive samples obtained from field isolates. The threshold for positivity for each species was determined using the mean value obtained from negative controls for each species, plus three times the standard deviation.
The primers and probes of the qPCR assay are listed in Table
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