Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood

Abstract

Monitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. The probes feature two strategic design elements: locked nucleic acids to enhance specificity and the reporter dyes Cy5 and TEX615, which have less optical overlap with the blood absorbance spectra than other commonly used dyes. Probe performance was validated on a traditional laboratory-based instrument and then further tested on a field-deployable Adaptive PCR instrument to develop a point-of-care platform appropriate for use in malaria settings. The probes discriminated between wild-type Plasmodium falciparum and the chloroquine-resistant CRT PF3D7_0709000:c.227A>C (p.Lys76Thr) mutant in the presence of 2% blood. Additionally, in allelic discrimination plots with the new probes, samples clustered more closely to their respective axes compared with samples using minor groove binder probes with 6-FAM and VIC reporter dyes. Our strategy greatly simplifies single-nucleotide polymorphism detection and provides a more accessible alternative for antimalarial resistance surveillance in the field.

Figure 1

Comparison of the minor groove binder (MGB) and locked nucleic acid (LNA) CRT c.227A>C probes in blood with a traditional PCR instrument. Each panel combines reactions with 0% to 3% whole blood. The results are shown as wild-type (solid line) or mutant (dashed line) oligonucleotide (oligo) of the CRT amplicon or a no-template control (dotted line). Each sample was performed in duplicate on the Rotor-Gene instrument. The normalized fluorescence intensity for the MGB (A and B) and the LNA (C and D) probe pairs is plotted for each cycle. In all cases, as the percentage of blood increases, the fluorescence intensity decreases. The FAM (A) and VIC (B) fluorescence intensities are more dramatically attenuated by the addition of blood compared with the Cy5 (C) and TEX615 (D) reporter dyes.

Figure 2

Performance of the locked nucleic acid CRT c.227A>C probes in blood with the Adaptive PCR instrument. The wild-type (A and B) or mutant (C and D) oligonucleotide or a no-template control (E and F) was amplified in the presence of 0%, 1%, 2%, or 3% whole blood on the Adaptive PCR device.

Figure 3

Allelic discrimination plots for each PCR instrument. The end point fluorescence values are plotted in allelic discrimination plots for the Rotor-Gene (A and B) and Adaptive PCR (C) instruments. For the former, minor groove binder (MGB; A) and locked nucleic acid (LNA; B) probes were tested, whereas LNA probes were evaluated in Adaptive PCR (C).

Figure 4

Single-nucleotide polymorphism genotyping from genomic Plasmodium falciparum DNA in the presence of 2% blood. Each Adaptive PCR sample was spiked with 2% blood and P. falciparum genomic DNA (gDNA) from either a chloroquine-sensitive/reference strain (3D7; solid red line) or a chloroquine-resistant strain (7G8; dashed red line). As a positive control, a subset of samples was spiked with the amplicon-length oligonucleotide (blue lines). The wild-type probe is labeled with Cy5 (A), and the mutant is labeled with TEX615 (B). NTC, no-template control.

Figure 5

The c.227A>C probes enable single-nucleotide polymorphism genotyping directly from Plasmodium falciparum–infected red blood cells (RBCs). Amplification curves for the wild-type (A) and mutant (B) probes. Washed, packed RBCs from a 3D7 (wild-type) culture were added to an Adaptive PCR sample at a final concentration of 0.8%. The same concentration of the supernatant after washing was also evaluated for the presence of parasite DNA. Uninfected packed RBCs and no-template controls served as negative controls.