An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.
An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.
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DOI：
10.1186/s12936-018-2362-x
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发表时间：
2018-05-29
期刊：
影响因子：
3
通讯作者：
Maeda T
中科院分区：
文献类型：
--
作者：
Imai K;Tarumoto N;Runtuwene LR;Sakai J;Hayashida K;Eshita Y;Maeda R;Tuda J;Ohno H;Murakami T;Maesaki S;Suzuki Y;Yamagishi J;Maeda T
抗青蒿素(抗逆转录病毒药物)恶性疟原虫最近的传播代表着对公共卫生的新的全球威胁。在东南亚，kelch13(K13)的C580Y突变是抗ART恶性疟原虫的显性突变。因此，迫切需要一种简单的方法来检测C580Y突变，以便在现场进行广泛的常规监测。本研究的目的是建立一种新的诊断C580Y突变的方法，将环介导的等温扩增(LAMP)与Minion纳米孔测序仪相结合。建立了检测恶性疟原虫K13基因C580Y突变的LAMP方法。该方法的检出限为60分钟内携带K13基因的参考质粒组分10份。此后，使用Minion纳米孔测序仪对LAMP产物进行扩增序列测定，以澄清该基因的核苷酸序列。根据测序开始30分钟后从Minion Reads中收集的序列数据，鉴定了C580Y突变。此外，对采集自印度尼西亚恶性疟疾患者的34份人类血液样本进行的LAMP检测的临床评估显示，阳性检测率为100%。使用Minion对多达12个样本的所有LAMP扩增片段进行了同时测序。测序结果与常规聚合酶链式反应和Sanger测序方法一致。从DNA提取到变异体调用在3小时内全部完成，在这34株印尼恶性疟原虫中未发现C580Y突变。一种结合LAMP和MINION的创新方法将能够简单、快速和高灵敏度地检测恶性疟原虫C580Y突变，即使在发展中国家资源有限的情况下也是如此。本文的在线版本(10.1186/s12936-0182362-x)包含补充材料，可供授权用户使用。
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 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. 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. The online version of this article (10.1186/s12936-018-2362-x) contains supplementary material, which is available to authorized users.
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DOI：
10.1093/infdis/jit184
发表时间：
2013-08-15
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
Hopkins H;González IJ;Polley SD;Angutoko P;Ategeka J;Asiimwe C;Agaba B;Kyabayinze DJ;Sutherland CJ;Perkins MD;Bell D
通讯作者：
Bell D
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10.4269/ajtmh.2012.12-0095
发表时间：
2013-02-01
影响因子：
3.3
作者：
Baltzell, Kimberly A.;Shakely, Deter;Greenhouse, Bryan
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Greenhouse, Bryan
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3.7
作者：
Lucchi NW;Demas A;Narayanan J;Sumari D;Kabanywanyi A;Kachur SP;Barnwell JW;Udhayakumar V
通讯作者：
Udhayakumar V
影响因子：
4.6
作者：
Imwong, Mallika;Jindakhad, Thantip;Dondorp, Arjen M.
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影响因子：
3.7
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Kyabayinze DJ;Zongo I;Cunningham J;Gatton M;Angutoko P;Ategeka J;Compaoré YD;Muehlenbachs A;Mulondo J;Nakalembe M;Somé FA;Ouattara A;Rouamba N;Ouédraogo JB;Hopkins H;Bell D
通讯作者：
Bell D