Imai Kazuo, Tarumoto Norihito, Misawa Kazuhisa, Runtuwene Lucky Ronald, Sakai Jun, Hayashida Kyoko, Eshita Yuki, Maeda Ryuichiro, Tuda Josef, Murakami Takashi, Maesaki Shigefumi, Suzuki Yutaka, Yamagishi Junya, Maeda Takuya
Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama, 359-8513, Japan.
Department of Infectious Disease and Infection Control, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan.
BMC Infect Dis. 2017 Sep 13;17(1):621. doi: 10.1186/s12879-017-2718-9.
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.
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 curtisi, P. 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.
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.
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.
由于传统显微镜检查存在局限性，迫切需要一种简单且准确的疟疾分子诊断方法。在本研究中，我们展示了一种使用环介导等温扩增（LAMP）和MinION™纳米孔测序仪诊断人类疟疾的新程序。
我们针对包括两个卵形疟原虫亚种在内的所有五种人类疟原虫的18S - rRNA基因生成了特异性LAMP引物（恶性疟原虫、间日疟原虫、沃氏卵形疟原虫、柯氏卵形疟原虫、诺氏疟原虫和三日疟原虫），并检测了从印度尼西亚63名疟疾患者采集的人类血液样本。此外，我们使用MinION™纳米孔测序仪对LAMP产物进行扩增子测序，以鉴定每种疟原虫物种。
我们的LAMP方法能够扩增参考质粒的所有靶向18S - rRNA基因，每个反应的检测限为10 - 100个拷贝。在63份临床样本中，分别有54份和55份样本通过巢式PCR和我们的LAMP方法检测为阳性。使用MinION™通过LAMP扩增子测序分析鉴定疟原虫物种，结果与参考质粒序列和巢式PCR结果一致。
我们结合LAMP和MinION™的诊断方法可能成为一种简单且准确的工具，用于鉴定人类疟原虫物种，即使在资源有限的情况下也是如此。
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