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Blood Drop Assay Designed for Monitoring Antimalarial Drug Resistance

By Labmedica International staff writers
Posted on 24 Jun 2019
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Image: A colorized electron micrograph showing malaria parasite (right, blue) attaching to a human red blood cell. The inset shows a detail of the attachment point at higher magnification (Photo courtesy of [U.S.]NIAID via Wikimedia Commons).
Image: A colorized electron micrograph showing malaria parasite (right, blue) attaching to a human red blood cell. The inset shows a detail of the attachment point at higher magnification (Photo courtesy of [U.S.]NIAID via Wikimedia Commons).
A new method for analyzing DNA directly in a drop of blood was designed for monitoring development of resistance to antimalarial drugs.

Monitoring of antimalarial resistance in the Plasmodium falciparum parasite is important to prevent further spread of the disease, but the available options for assessing resistance are not usually applicable to field conditions. 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 an approach more suited for use outside the traditional laboratory, investigators at Vanderbilt University (Nashville, TN, USA) designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enabled single-nucleotide polymorphism genotyping directly from whole blood. The Taq polymerase enzyme is a thermostable DNA polymerase I named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

The new probes featured 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.

Adaptive PCR is a previously described real-time PCR platform that used left-handed DNA (L-DNA) additives to monitor the reaction for more reliable point-of-care performance. This method was fundamentally simpler and more robust than traditional PCR. It functions by dynamically controlling thermal cycling through direct monitoring of the two key hybridization events - primer annealing and product melting - during the reaction.

Results obtained during the study revealed that the probes could discriminate between wild-type P. falciparum and a chloroquine-resistant mutant in the presence of 2% blood. This strategy greatly simplified single-nucleotide polymorphism detection and provided a more accessible alternative for antimalarial resistance surveillance in the field.

"To mitigate the inhibition by blood components, we redesigned the molecular tools used for DNA analysis. We utilized reporter dyes that are more optically compatible with blood, which were combined with a specific type of DNA subunit to accurately pinpoint mutations. The end result is an assay in which blood is directly added to a reaction tube to detect mutations associated with antimalarial drug resistance," said senior author Dr. Frederick R. Haselton, professor of biomedical engineering and chemistry at Vanderbilt University.

The antimalarial resistance study was published in the June 13, 2019, online edition of the Journal of Molecular Diagnostics.

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Vanderbilt University


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