Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax |
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| Institution: | 1. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, 111 Phahonyothin Road, Klong Nueng, Klong Luang, Pathum Thani 12120, Thailand;2. Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand;1. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phahonyothin Rd., Klong Neung, Klong Luang, Pathum Thani 12120, Thailand;2. CENTEX Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand;3. Bioengineering and Sensing Technology Laboratory, BIOTEC, NSTDA,113 Thailand Science Park, Phahonyothin Rd., Klong Neung, Klong Luang, Pathum Thani 12120, Thailand;4. Shrimp-virus interaction laboratory (ASVI), BIOTEC, NSTDA, Yothi office, Rama VI Rd.,Bangkok 10400, Thailand;1. Department of Microbiology, Immunology and Infectious Disease, University of Calgary, AB, Canada;2. Department of Pathology and Laboratory Medicine, University of Calgary, AB, Canada;4. Department of Medicine, University of Calgary, Calgary, AB, Canada |
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| Abstract: | 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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