title: Application of loop-mediated isothermal amplification assay combined with lateral flow dipstick for detection of Plasmodium falciparum and Plasmodium vivax.
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 1.5h. 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.
authors.affiliation: 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.
authors.forename: Suganya
authors.initials: S
authors.lastname: Yongkiettrakul
authors.affiliation: Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand.
authors.forename: Wansadaj
authors.initials: W
authors.lastname: Jaroenram
authors.affiliation: 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; Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand.
authors.forename: Narong
authors.initials: N
authors.lastname: Arunrut
authors.affiliation: 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.
authors.forename: Wanwisa
authors.initials: W
authors.lastname: Chareanchim
authors.affiliation: 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.
authors.forename: Supicha
authors.initials: S
authors.lastname: Pannengpetch
authors.affiliation: 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; Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand.
authors.forename: Rungkarn
authors.initials: R
authors.lastname: Suebsing
authors.affiliation: 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; Center of Excellence for Shrimp Molecular Biology and Biotechnology (CENTEX Shrimp), Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand.
authors.forename: Wansika
authors.initials: W
authors.lastname: Kiatpathomchai
authors.affiliation: 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.
authors.forename: Wichai
authors.initials: W
authors.lastname: Pornthanakasem
authors.affiliation: 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.
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.affiliation: 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. Electronic address: darin@biotec.or.th.
authors.forename: Darin
authors.initials: D
authors.lastname: Kongkasuriyachai
mesh_terms: D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology*; D016780:Malaria, Vivax / Q000469:parasitology*; D009097:Multienzyme Complexes / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010963:Plasmodium falciparum / Q000235:genetics / Q000302:isolation & purification*; D010966:Plasmodium vivax / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D009097:Multienzyme Complexes; D015800:Protozoan Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase
keywords: Lateral flow dipstick; Loop-mediated isothermal amplification; Plasmodium falciparum; Plasmodium vivax