A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION (TM) nanopore sequencer

作者：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^*

来源：BMC Infectious Diseases, 2017, 17(1): 621.

DOI：10.1186/s12879-017-2718-9

• Malaria
• Plasmodium ovale curtisi
• Plasmodium ovale wallikeri
• LAMP
• Nanopore sequencer
• MinION (TM)
• Diagnosis

Background: 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 (TM) nanopore sequencer.
Methods: 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 (TM) nanopore sequencer to identify each Plasmodium species.
Results: 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 (TM) was consistent with the reference plasmid sequences and the results of nested PCR.
Conclusions: Our diagnostic method combined with LAMP and MinION (TM) could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

• 出版日期2017-9-13