Luna universal probe qpcr master mix neb

Quantitative PCR (qPCR) was performed using a real-time PCR system (QuantStudio TM 5) to detect and quantify the Plasmodium falciparum in duplicate. Primers and probes were based on the 18S rRNA sequence [27] synthesised from Inqaba Biotec Laboratory, South Africa. The amplification followed a method in a previous study [28] . The CT values were recorded and used to calculate parasite clearance through fold change, 2 -ΔΔCT by a comparative CT value method [29] . The method considered two data points of CT values at day 0 and day 3 for individuals that remained positive on day 3. Specific primers were used for the qPCR (Table 1); the probe was labelled with 5′FAM (6-carboxyfluorescein) and BHQ-1 TM (Black Hole Quencher) as the reporter and quencher, respectively. 20 μl reaction volume was prepared, composed of 0.8 μl of each forward and reverse primer, 10 μl of qPCR master mix (Luna Universal Probe qPCR Master Mix NEB, New England BioLabs Inc), 0.4 μl of the probes, 20 ng of the DNA template and nuclease-free water (variable). The PCR conditions involved one cycle at 94°C for 5 min of initial denaturation, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 90 s and extension at 68°C for 90 s, and a final extension at 68°C for 90 s.