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

Publication

Date: 13th September 2017 | Source: BMC Infectious Diseases

Authors: Kazuo Imai, Norihito Tarumoto, Kazuhisa Misawa, Lucky Ronald Runtuwene, Jun Sakai, Kyoko Hayashida, Yuki Eshita, Ryuichiro Maeda, Josef Tuda, Takashi Murakami, Shigefumi Maesaki, Yutaka Suzuki, Junya Yamagishi, Takuya Maeda.

Background

A 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.

Methods

We 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 curtisiP. 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.

Results

Our 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.

Conclusions

Our 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.

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