Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax

•We developed LAMP protocols for species detection of malaria dhfr-ts gene.•LAMP combined with simple strip-test provided a user-friendly format within 1.5 h.•The malaria dhfr-ts LAMP protocol requires minimal sample preparation steps.

Malaria is largely a preventable and curable disease. However, a delay or an inappropriate treatment can result in serious adverse outcomes for patient. Rapid, simple and cost-effective diagnostic tests that can be easily adapted and rapidly scaled-up at the field or community levels are needed. In this study, accelerated detection methods for the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) dihydrofolate reductase-thymidylate synthase were developed based on the loop-mediated isothermal amplification (LAMP) method. The developed methods included the use of species-specific biotinylated primers to amplify LAMP amplicons, which were then hybridized to specific FITC-labeled DNA probes and visualized on a chromatographic lateral flow dipstick (LFD). The total LAMP-LFD assay time was approximately 1.5 h. The LAMP-LFD assays showed similar detection limit to conventional PCR assay when performed on plasmid DNA carrying the malaria dhfr-ts genes. The LAMP-LFD showed 10 folds higher detection limit than PCR when performed on genomic DNA samples from Pf and Pv parasites. The dhfr-ts LAMP-LFD assays also have the advantages of reduced assay time and easy format for interpretation of results.

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