Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing

Hayashida, Kyoko; Orba, Yasuko; Sequeira, Patricia C.; Sugimoto, Chihiro; Hall, William W.; Eshita, Yuki; Suzuki, Yutaka; Runtuwene, Lucky; Brasil, Patricia; Calvet, Guilherme; Rodrigues, Cintia D. S.; dos Santos, Carolina C.; Mares-Guia, Maria A. M.; Yamagishi, Junya; de Filippis, Ana M. B.; Sawa, Hirofumi

Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing

Hayashida, Kyoko; Orba, Yasuko; Sequeira, Patricia C. et al.

PLoS neglected tropical diseases, 2019, 13(6), e0007480

Permalink : https://hdl.handle.net/2115/75177

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アクセス権	open access
URI	https://hdl.handle.net/2115/75177
タイトル	Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing
著者	Hayashida, Kyoko ; Orba, Yasuko ; Sequeira, Patricia C. ; Sugimoto, Chihiro ; Hall, William W. ; Eshita, Yuki ; Suzuki, Yutaka ; Runtuwene, Lucky ; Brasil, Patricia ; Calvet, Guilherme ; Rodrigues, Cintia D. S. ; dos Santos, Carolina C. ; Mares-Guia, Maria A. M. ; Yamagishi, Junya ; de Filippis, Ana M. B. ; Sawa, Hirofumi
著者名 Hayashida, Kyoko
e-Rad_Researcher 60507169 著者名 Orba, Yasuko
著者名 Sequeira, Patricia C.
e-Rad_Researcher 90231373 著者名 Sugimoto, Chihiro
著者名 Hall, William W.
e-Rad_Researcher 10082223 著者名 Eshita, Yuki
e-Rad_Researcher 40323646 著者名 Suzuki, Yutaka
e-Rad_Researcher 40793782 著者名 Runtuwene, Lucky
著者名 Brasil, Patricia
著者名 Calvet, Guilherme
著者名 Rodrigues, Cintia D. S.
著者名 dos Santos, Carolina C.
著者名 Mares-Guia, Maria A. M.
e-Rad_Researcher 80535328 著者名 Yamagishi, Junya
著者名 de Filippis, Ana M. B.
e-Rad_Researcher 30292006 著者名 Sawa, Hirofumi
言語	英語
発行日	2019-06-03
出版者	PLOS
収録物名	PLoS neglected tropical diseases
巻(号)	13(6)
ページ	e0007480
抄録	Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas. Author summary Chikungunya virus has re-emerged as an important pathogen causing several outbreaks in the world. As the clinical symptoms of chikungunya is similar to other mosquito-borne febrile diseases, the definitive diagnosis of the disease is based on the detection of viral genome from the patient blood. Loop-mediated isothermal amplification (LAMP) is a method that rapidly amplify nucleic acids under isothermal condition. In the present work, a simple dried format LAMP test for chikungunya diagnosis was developed which can be directly amplified from human blood. Combining with the portable sequencer MinION sequencing system, a method to identify the viral genotype was also established. The developed on-site diagnosis and genotyping system is easy to perform, sensitive, and rapid. Therefore, it offers great promise as a routine simple tool for diagnosis and disease management of chikungunya.
資源タイプ	学術雑誌論文
出版タイプ	Version of Record
権利情報	Creative Commons Attribution 4.0 International
PISSN	1935-2727
EISSN	1935-2735
関連情報	PMID 31158221
関連情報 (isIdenticalTo)	出版社版DOI https://doi.org/10.1371/journal.pntd.0007480