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

• Published in: Parasitology international 2014-12, Vol.63 (6), p.777-784
• Main Authors: Yongkiettrakul, Suganya, Jaroenram, Wansadaj, Arunrut, Narong, Chareanchim, Wanwisa, Pannengpetch, Supicha, Suebsing, Rungkarn, Kiatpathomchai, Wansika, Pornthanakasem, Wichai, Yuthavong, Yongyuth, Kongkasuriyachai, Darin
• Format: Article
• Language: English
• Subjects: DNA Primers - genetics; DNA, Protozoan - genetics; Gastroenterology and Hepatology; Humans; Infectious Disease; Lateral flow dipstick; Loop-mediated isothermal amplification; Malaria, Falciparum - parasitology; Malaria, Vivax - parasitology; Multienzyme Complexes - genetics; Nucleic Acid Amplification Techniques - methods; Plasmodium falciparum; Plasmodium falciparum - genetics; Plasmodium falciparum - isolation & purification; Plasmodium vivax; Plasmodium vivax - genetics; Plasmodium vivax - isolation & purification; Polymerase Chain Reaction - methods; Protozoan Proteins - genetics; Sensitivity and Specificity; Tetrahydrofolate Dehydrogenase - genetics; Thymidylate Synthase - genetics
• ISSN: 1383-5769; 1873-0329
• DOI: 10.1016/j.parint.2014.06.004

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.