An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer

Malaria Journal
Kazuo ImaiTakuya Maeda

Abstract

The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. 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. A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 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 P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP ...Continue Reading

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Sep 21, 2018·PloS One·Christian NsanzabanaIveth J Gonzalez
Dec 11, 2019·Tropical Medicine and Infectious Disease·Christian Nsanzabana
Oct 5, 2019·Journal of Human Genetics·Arthur Elia MonganLucky Ronald Runtuwene
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Mar 9, 2021·The Korean Journal of Parasitology·Thunchanok KhammaneeSupinya Thanapongpichat
Aug 2, 2021·International Journal for Parasitology. Drugs and Drug Resistance·Yaye Die NdiayeSarah K Volkman

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Datasets Mentioned

BETA
KT956001.1
KR537460.1

Methods Mentioned

BETA
genotyping
nucleic
PCR
amplicon sequencing
Ligation Sequencing

Software Mentioned

BCFtools
Poretools
Porechop
MinKNOW
PrimerExplorer
MEM
Samtools
MinION
BWA
ONT

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