title: Loop-Mediated Isothermal Amplification and LFD Combination for Detection of Plasmodium falciparum and Plasmodium vivax.
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.
authors.affiliation: National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand. darin@biotec.or.th.
authors.forename: Darin
authors.initials: D
authors.lastname: Kongkasuriyachai
authors.affiliation: National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
authors.forename: Suganya
authors.initials: S
authors.lastname: Yongkiettrakul
authors.affiliation: National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
authors.affiliation: National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.
authors.forename: Narong
authors.initials: N
authors.lastname: Arunrut
mesh_terms: D017931:DNA Primers; D016054:DNA, Protozoan; D017125:Genes, Protozoan; D006801:Humans; D008288:Malaria / Q000175:diagnosis; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics*; D010966:Plasmodium vivax / Q000235:genetics*
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan
keywords: Detection; LAMP; LFD; Lateral flow dipstick; Loop-mediated isothermal amplification; Malaria; Plasmodium