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

BMC Infectious Diseases

Research Article

Takashi Murakami¹  Takuya Maeda¹  Lucky Ronald Runtuwene²  Yutaka Suzuki²  Norihito Tarumoto³  Jun Sakai³  Shigefumi Maesaki³  Josef Tuda⁴  Ryuichiro Maeda⁵  Kazuo Imai⁶  Kazuhisa Misawa⁶  Kyoko Hayashida⁷  Yuki Eshita⁸  Junya Yamagishi⁹ 

[1] Center for Clinical Infectious Diseases and Research, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, 350-0495, Saitama, Japan;Department of Microbiology, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, 350-0495, Saitama, Japan;Department of Computational Biology and Medical Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, 277-8562, Chiba, Japan;Department of Infectious Disease and Infection Control, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, 350-0495, Saitama, Japan;Center for Clinical Infectious Diseases and Research, Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, 350-0495, Saitama, Japan;Department of Parasitology, Faculty of Medicine, Sam Ratulangi University, Kampus Unsrat, 95115, Manado, Bahu, Indonesia;Division of Biomedical Sciences, Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Inada-cho, 080-8555, Obihiro, Hokkaido, Japan;Division of Infectious Diseases and Pulmonary Medicine, Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, 359-8513, Saitama, Japan;Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, 001-0020, Sapporo, Hokkaido, Japan;Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, 001-0020, Sapporo, Hokkaido, Japan;Faculty of Medicine, Oita University, 1-1 Hasama-machi, 879-5593, Yufu, Oita, Japan;Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Thung Phaya, Ratchathewi, 10400, Bangkok, Thailand;Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565-0871, Osaka, Japan;Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, 001-0020, Sapporo, Hokkaido, Japan;Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, North 20, West 10 Kita-ku, 001-0020, Sapporo, Hokkaido, Japan;

关键词: Malaria;    Plasmodium ovale curtisi;    Plasmodium ovale wallikeri;    LAMP;    Nanopore sequencer;    MinION™;    Diagnosis;   

DOI  :  10.1186/s12879-017-2718-9

 received in 2017-02-20, accepted in 2017-09-06,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundA 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.MethodsWe 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.ResultsOur 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.ConclusionsOur 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.

【 授权许可】

CC BY   
© The Author(s). 2017