Abstract
Loop-mediated isothermal amplification (LAMP) has been used to detect several pathogens including malaria parasites from field and clinical samples. In this protocol, the malaria LAMP technology is developed to differentiate between Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) species by targeting the dihydrofolate reductase thymidylate synthase (dhfr-ts) gene, a known target for the antifolate class of drugs such as Pyrimethamine. LAMP primer sets are designed and validated for species specific amplification. Additionally, specific probes help improve detection and visualization of the products when combined with lateral flow dipstick-based (LFD) detection. The protocols are further simplified to eliminate tedious sample preparation steps, such that crude lysis prepared simply by diluting few microliter (μL) of blood sample with distilled water is sufficient. The LAMP-LFD malaria dhfr-ts protocols are sensitive and can detect as little as 1 picogram (pg) of PfDNA and 1 nanogram (ng) of PvDNA, or a few microliters of crude lysate from infected blood samples (Yongkiettrakul et al., Parasitol Int 63: 777–784, 2014). These simplified steps not only reduce cost but also increase the potential for large application in the fields and clinical settings.
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Acknowledgments
This research was sponsored by Research Director’s Initiative Fund, National Center for Genetic Engineering and Biotechnology, National Center for Science and Development Agency, Thailand.
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Kongkasuriyachai, D., Yongkiettrakul, S., Kiatpathomchai, W., Arunrut, N. (2017). Loop-Mediated Isothermal Amplification and LFD Combination for Detection of Plasmodium falciparum and Plasmodium vivax . In: Prickril, B., Rasooly, A. (eds) Biosensors and Biodetection. Methods in Molecular Biology, vol 1572. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6911-1_28
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DOI: https://doi.org/10.1007/978-1-4939-6911-1_28
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