Ultra-sensitive qPCR Detection of Plasmodium falciparum

DNA was extracted from 100 μL blood using the Genomic DNA Extraction kit (Macherey-Nagel, Düren, Germany) and eluted in an equivalent volume of elution buffer. 4 μL of DNA, corresponding to 4 μL of blood was screened for P. falciparum using ultrasensitive qPCR that amplifies a conserved region of the var gene acidic terminal sequence (varATS) according to a previously published protocol [48 (link)]. The varATS gene assay amplifies ~20 copies/genome [48 (link)]. Absolute parasite densities were obtained using an external standard curve of ten-fold serial dilutions (5-steps) of 3D7 P. falciparum parasites quantified by droplet digital PCR (ddPCR) [49 (link)]. The ddPCR thermocycling conditions, sequences and concentration of primers and probe are given in an additional file [50 (link)].

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Oduma C.O., Ombok M., Zhao X., Huwe T., Ondigo B.N., Kazura J.W., Grieco J., Achee N., Liu F., Ochomo E, & Koepfli C. (2023). Altitude, not potential larval habitat availability, explains pronounced variation in Plasmodium falciparum infection prevalence in the western Kenya highlands. PLOS Global Public Health, 3(4), e0001505.

Publication 2023

Genomic DNA Extraction kit

Acidic Assay Blood Buffer Conserved sequence Dilutions Gene Genome Parasites Primers

Corresponding Organization : University of Notre Dame

Other organizations : Kenya Medical Research Institute, Egerton University, Case Western Reserve University, Center for Global Health

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Absolute parasite densities of P. falciparum

control variables

DNA extraction procedure (Genomic DNA Extraction kit, Macherey-Nagel, Düren, Germany)
Elution volume (equivalent to the initial blood volume of 100 μL)
QPCR assay (ultrasensitive qPCR that amplifies a conserved region of the varATS gene)
External standard curve (ten-fold serial dilutions of 3D7 P. falciparum parasites quantified by droplet digital PCR)

controls

Positive control: 3D7 P. falciparum parasites quantified by droplet digital PCR
Negative control: Not explicitly mentioned

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