An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer
© 2018 The Author(s). Background: 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 f...
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th-mahidol.460142019-08-28T13:08:31Z An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer Kazuo Imai Norihito Tarumoto Lucky Ronald Runtuwene Jun Sakai Kyoko Hayashida Yuki Eshita Ryuichiro Maeda Josef Tuda Hideaki Ohno Takashi Murakami Shigefumi Maesaki Yutaka Suzuki Junya Yamagishi Takuya Maeda Sam Ratulangi University Oita University University of Tokyo Osaka University Saitama Medical University Hokkaido University Saitama Medical Center, Saitama Medical University Mahidol University Obihiro University of Agriculture and Veterinary Medicine Immunology and Microbiology Medicine © 2018 The Author(s). Background: 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. Results: 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 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 P. falciparum isolates in Indonesia. Conclusions: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries. 2019-08-23T11:20:41Z 2019-08-23T11:20:41Z 2018-05-29 Article Malaria Journal. Vol.17, No.1 (2018) 10.1186/s12936-018-2362-x 14752875 2-s2.0-85047728996 https://repository.li.mahidol.ac.th/handle/123456789/46014 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85047728996&origin=inward |
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Immunology and Microbiology Medicine Kazuo Imai Norihito Tarumoto Lucky Ronald Runtuwene Jun Sakai Kyoko Hayashida Yuki Eshita Ryuichiro Maeda Josef Tuda Hideaki Ohno Takashi Murakami Shigefumi Maesaki Yutaka Suzuki Junya Yamagishi Takuya Maeda An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer |
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© 2018 The Author(s). Background: 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. Results: 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 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 P. falciparum isolates in Indonesia. Conclusions: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries. |
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Sam Ratulangi University |
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Sam Ratulangi University Kazuo Imai Norihito Tarumoto Lucky Ronald Runtuwene Jun Sakai Kyoko Hayashida Yuki Eshita Ryuichiro Maeda Josef Tuda Hideaki Ohno Takashi Murakami Shigefumi Maesaki Yutaka Suzuki Junya Yamagishi Takuya Maeda |
format |
Article |
author |
Kazuo Imai Norihito Tarumoto Lucky Ronald Runtuwene Jun Sakai Kyoko Hayashida Yuki Eshita Ryuichiro Maeda Josef Tuda Hideaki Ohno Takashi Murakami Shigefumi Maesaki Yutaka Suzuki Junya Yamagishi Takuya Maeda |
author_sort |
Kazuo Imai |
title |
An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer |
title_short |
An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer |
title_full |
An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer |
title_fullStr |
An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer |
title_full_unstemmed |
An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer |
title_sort |
innovative diagnostic technology for the codon mutation c580y in kelch13 of plasmodium falciparum with minion nanopore sequencer |
publishDate |
2019 |
url |
https://repository.li.mahidol.ac.th/handle/123456789/46014 |
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1763491259915173888 |