A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer

Abstract

BackgroundA simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer.MethodsWe generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species.ResultsOur LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR.ConclusionsOur diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.Electronic supplementary materialThe online version of this article (10.1186/s12879-017-2718-9) contains supplementary material, which is available to authorized users.

Nanopore sequencing: Review of potential applications in functional genomics

“…For these purposes, mobile lab approaches for DNA extraction and library preparation have been explored using devices like VolTRAX V2 or Bento Lab (all-in-one DNA laboratory including thermal cycler: https://www.bento.bio), and experimental protocol developments are also underway, such as those using LAMP (loop-mediated isothermal amplification) methods instead of conventional PCR to eliminate the need for a thermal cycler. LAMP only requires a water bath to conduct its isothermal amplification, and Imai and colleagues demonstrated an identification system for human Plasmodium species (Imai et al, 2017). Yamagishi and colleagues also reported the serotyping system for dengue virus in a single day (Yamagishi et al, 2017) (Greninger et al, 2015).…”

Size and sequence polymorphisms in the glutamate-rich protein gene of the human malaria parasite Plasmodium falciparum in Thailand

“…A number of PCR-based diagnostic assays have been developed and increasingly adopted for clinical malaria diagnostics [ 1 , 2 ]. For identification of malaria species, house-keeping genes have been chosen as molecular targets for developing PCR assays, including the small subunit ribosomal RNA gene in the nuclear genome as well as the cytochrome c oxidase subunit 1 gene in the mitochondrial genome [ 3 – 6 ]. Additionally, PCR-based assays can be performed using polymorphic loci, including amplified fragment length polymorphism, microsatellites and antigen-coding genes, facilitating the identification of the parasite strains, the identification of monoclonal vs polyclonal infections, and the differentiation between relapse vs re-infections [ 7 – 11 ].…”

Allele-Specific Isothermal Amplification Method Using Unmodified Self-Stabilizing Competitive Primers

“…Other reported isothermal assays usually need between 20 to 75 min to finish, 45 , 46 do not report quantitative data, 47 rely on the addition of probes (Au NPs) to enhance specificity, 48 lack amplification data at low concentrations, 26 or require sequencing for product specificity. 49 …”