Abstract
Malaria is among the major threats to global health with the main burden of disease being in rural areas of developing countries where accurate diagnosis based on non-invasive samples is in high demand. We here present a novel molecular assay for detection of malaria parasites based on technology that may be adapted for low-resource settings. Moreover, we demonstrate the exploitation of this assay for detection of malaria in saliva. The setup relies on pump-free microfluidics enabled extraction combined with a DNA sensor substrate that is converted to a single-stranded DNA circle specifically by topoisomerase I expressed by the malaria causing Plasmodium parasite. Subsequent rolling circle amplification of the generated DNA circle in the presence of biotin conjugated deoxynucleotides resulted in long tandem repeat products that was visualized colorimetrically upon binding of horse radish peroxidase (HRP) and addition of 3,3′,5,5′-Tetramethylbenzidine that was converted to a blue colored product by HRP. The assay was directly quantitative, specific for Plasmodium parasites, and allowed detection of Plasmodium infection in a single drop of saliva from 35 out of 35 infected individuals tested. The results could be determined directly by the naked eye and documented by quantifying the color intensity using a standard paper scanner.
Highlights
Malaria is a widespread tropical disease causing approximately half a million deaths worldwide
The generated DNA circles where amplified by rolling circle amplification (RCA) and the products hybridized with fluorescent labeled probes before they where visualized at the single molecule level in a fluorescence microscope resulting in a detection limit of 0.06 parasites/μL using only a single drop of blood[34]
Specific REEAD assay, which measures the activity of the essential Plasmodium expressed pTopI enzyme, and the combination of this assay with droplet microfluidic assisted sample extraction (REEAD-on-a-chip) has previously been described for specific and highly sensitive detection of Plasmodium parasites in blood samples from malaria positive individuals[34]
Summary
Malaria is a widespread tropical disease causing approximately half a million deaths worldwide. Combined with a specialized microfluidics based extraction system RCA enhanced enzyme activity detection (in short REEAD) was previously exploited for highly sensitive detection of the malaria causing Plasmodium parasites[34] This was achieved using a DNA sensor module that was circularized by the DNA cleavage-ligation activity of the Plasmodium specific enzyme, topoisomerase I (pTopI). The generated DNA circles where amplified by RCA and the products hybridized with fluorescent labeled probes before they where visualized at the single molecule level in a fluorescence microscope resulting in a detection limit of 0.06 parasites/μL using only a single drop of blood[34] This sensitivity of pTopI specific REEAD was superior to the already developed gold standard diagnostic methods[47,48] and better than most of the reported PCR protocols[49] These findings suggest that the REEAD technology has the potential for being further developed into a highly sensitive diagnostic test. The possibility of using a noninvasive sample type such as saliva as test material for the pTopI based assay holds great promise for the future, since the REEAD technology, once fully developed, may be used for diagnosis in areas with cultural reluctance of giving blood as well as for eradication programs, which may involve testing of large number of asymptomatic individuals
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