Constructing a molecular assay based on the recombinase polymerase amplification technique for simultaneous detection of Plasmodium falciparum and Plasmodium vivax

Abstract

Malaria is an infectious disease caused by Plasmodium species. In Vietnam, this disease is mainly caused by Plasmodium falciparum and Plasmodium vivax through the malaria vector, the Anopheles mosquito. Malaria mainly circulates throughout the year in forest, mountainous, and coastal areas in Vietnam where diagnostic methods for this disease are often not easily accessible. Vaccines for malaria have been not available, but malaria medications are effective against the disease especially when malaria is diagnosed at the early stage. In this study, we developed a molecular assay based on the recombinase polymerase amplification (RPA) technique to detect P. falciparum and P. vivax in clinical samples. We successfully designed the specific primers to amplify the DNA of these two Plasmodium species in the duplex RPA reaction. Reaction temperature and reaction volume of the duplex RPA reaction were studied to reduce the operating cost. We also set up the hybridisation reaction with the specific P. falciparum and P. vivax probes based on the lateral flow strip technique to detect the RPA products. This hybridisation technique reduced manipulation and timing as well as improved the accuracy in the detection of P. falciparum and P. vivax by RPA. Finally, we evaluated the built molecular assay to detect P. falciparum and P. vivax on 33 DNA samples collected from malaria patients in comparison with the monoplex real-time PCR assay. The results of the two assays were identical. The duplex RPA assay could be developed into a quick test kit for the rapid and accuracy detection of P. falciparum and P. vivax in the treatment of malaria in Vietnam.