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- Title
An innovative diagnostic technology for the codon mutation C580Y in <italic>kelch13</italic> of <italic>Plasmodium falciparum</italic> with MinION nanopore sequencer.
- Authors
Imai, Kazuo; Tarumoto, Norihito; Runtuwene, Lucky Ronald; Sakai, Jun; Hayashida, Kyoko; Eshita, Yuki; Maeda, Ryuichiro; Tuda, Josef; Ohno, Hideaki; Murakami, Takashi; Maesaki, Shigefumi; Suzuki, Yutaka; Yamagishi, Junya; Maeda, Takuya
- Abstract
Background: The recent spread of artemisinin (ART)-resistant <italic>Plasmodium falciparum</italic> represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of <italic>kelch13</italic> (<italic>k13</italic>) is the dominant mutation of ART-resistant <italic>P. falciparum.</italic> Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. Results: A LAMP assay for the <italic>k13</italic> gene of <italic>P. falciparum</italic> to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the <italic>k13</italic> gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with <italic>P. falciparum</italic> malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 <italic>P. falciparum</italic> isolates in Indonesia. Conclusions: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of <italic>P. falciparum</italic>, even in resource-limited situations in developing countries.
- Subjects
PLASMODIUM falciparum; ARTEMISININ; GENETIC mutation; NUCLEOTIDE sequencing; DRUG resistance
- Publication
Malaria Journal, 2018, Vol 17, Issue 1, pN.PAG
- ISSN
1475-2875
- Publication type
Article
- DOI
10.1186/s12936-018-2362-x