A powerful qPCR-high resolution melting assay with taqman probe in plasmodium species differentiation

BackgroundThe use of highly sensitive molecular tools in malaria diagnosis is currently largely restricted to research and epidemiological settings, but will ultimately be essential during elimination and potentially eradication. Accurate diagnosis and differentiation down to species levels, including the two Plasmodium ovale species and zoonotic variants of the disease, will be important for the understanding of changing epidemiological patterns of the disease.MethodsA qPCR-high resolution melting (HRM) method was to detect and differentiate all human Plasmodium species with one forward and one reverse primer set. The HRM detection method was further refined using a hydrolysis probe to specifically discriminate Plasmodium falciparum.ResultsOut of the 113 samples tested with the developed HRM-qPCR- P. falciparum probe assay, 96 (85.0%) single infections, 12 (10.6%) mixed infections, and 5 (4.4%) were Plasmodium negative. The results were concordant with those of the nested PCR at 98.2%. The assay limit of detection was varied from 21.47 to 46.43 copies /mu l, equivalent to 1-2.11 parasites/mu l. All P. falciparum infections were confirmed with the associated Taqman probe.ConclusionsAlthough the dependence on qPCR currently limits its deployment in resource-limited environments, this assay is highly sensitive and specific, easy to perform and convenient for Plasmodium mono-infection and may provide a novel tool for rapid and accurate malaria diagnosis also in epidemiological studies.

Automated summary

The development of a qPCR-HRM detection method for all human Plasmodium species, with high sensitivity and specificity, provides a novel tool for rapid and accurate malaria diagnosis, particularly for mono-infections. The method still faces limitations in resource-limited environments, but shows potential for use in research and epidemiological studies.