A Novel Diagnostic Method for Malaria Using Loop Mediated Isothermal Amplification (LAMP) and MinION Nanopore Sequencer

Abstract

BackgroundSimply and accurately diagnostic tool for Malaria is required for clinical diagnosis and epidemiological survey. We have developed a novel diagnostic tool for Malaria using loop mediated isothermal amplification (LAMP) with MinION nanopore sequencer.MethodsIn this study, we have designed human Plasmodium parasites-specific LAMP primers targeting for the lesion of 18S rDNA gene, which were locating on the conserved sequences across all five Plasmodium species; Plasmodium falciparum, P. vivax, P. ovale (P.o. wallikeri and P.o crutisi), P. knowlesi and P. malariae, containing each species-specific sequence within F1-B1 primer pairs. The sensitivities were evaluated using 10-fold serially diluted plasmids harboring the sequences of 18S rDNA. We also applied our protocol to human blood samples collected and stored with FTA elute cards derived from 30 Malaria patients, who are clinically diagnosed as Malaria in Indonesia. Its analytical sensitivities and specificities were also evaluated while comparing the results of previously described nested PCR methods. Finally, we performed amplicon sequencing of our LAMP methods using MinION nanopore sequencer to identify each Plasmodium species.ResultsOur LAMP method could amplify all targeting 18S rDNA gene on constructed plasmids and its detection limits were 10 - 100 copies/reaction respectively. In clinical samples, obtained LAMP results were completely consistent with the results of nested PCR. Additionally, identifications of Plasmodium species based on the sequence analysis with MinION were also consistent with the sequence of each constructed plasmid and could consistently confirmed its Plasmodium species with the highest homology of reference Plasmodiumparasite sequence.ConclusionOur innovative diagnostic technology with LAMP and MinION could become a powerful tool for identification of Plasmodium parasites even in resource-limited situation.Disclosures All authors: No reported disclosures.