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FIELD DIAGNOSIS AND GENOTYPING OF CHIKUNGUNYA VIRUS USING A DRIED REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) ASSAY AND MINION SEQUENCING
Hayashida, Kyoko et al. | Date Issued: 2019
Author
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.
Santos, Carolina C. dos
Mares-Guia, Maria A. M.
Yamagishi, Junya
Filippis, Ana M. B. de
Sawa, Hirofumi
Affilliation
Research Center for Zoonosis Control. Hokkaido University. Sapporo, Japan.
Research Center for Zoonosis Control. Hokkaido University. Sapporo, Japan.
Oswaldo Cruz Foundation. Oswaldo Cruz Institute. Flavivirus Laboratory. Rio de Janeiro, RJ, Brazil.
Research Center for Zoonosis Control. Hokkaido University. Sapporo, Japan / Global Station for Zoonosis Control. Global Institution for Collaborative Research and Education (GI-CoRE). Hokkaido University. Sapporo, Japan.
Global Station for Zoonosis Control. Global Institution for Collaborative Research and Education (GI-CoRE). Hokkaido University. Sapporo, Japan / Centre for Research in Infectious Diseases. School of Medicine and Medical Science. University College Dublin. Dublin, Ireland / Global Virus Network. Baltimore, Maryland, USA.
Research Center for Zoonosis Control. Hokkaido University. Sapporo, Japan.
Global Virus Network. Baltimore, Maryland, USA.
Global Virus Network. Baltimore, Maryland, USA.
Oswaldo Cruz Foundation. Evandro Chagas National Institute of Infectious Diseases. Acute Febrile Illnesses Laboratory. Rio de Janeiro, RJ, Brazil / Global Virus Network. Baltimore, Maryland, USA.
Oswaldo Cruz Foundation. Evandro Chagas National Institute of Infectious Diseases. Acute Febrile Illnesses Laboratory. Rio de Janeiro, RJ, Brazil / Global Virus Network. Baltimore, Maryland, USA.
Oswaldo Cruz Foundation. Oswaldo Cruz Institute. Flavivirus Laboratory. Rio de Janeiro, RJ, Brazil.
Oswaldo Cruz Foundation. Oswaldo Cruz Institute. Flavivirus Laboratory. Rio de Janeiro, RJ, Brazil.
Oswaldo Cruz Foundation. Oswaldo Cruz Institute. Flavivirus Laboratory. Rio de Janeiro, RJ, Brazil.
Research Center for Zoonosis Control. Hokkaido University. Sapporo, Japan / Global Station for Zoonosis Control. Global Institution for Collaborative Research and Education (GI-CoRE). Hokkaido University. Sapporo, Japan.
Oswaldo Cruz Foundation. Oswaldo Cruz Institute. Flavivirus Laboratory. Rio de Janeiro, RJ, Brazil.
Research Center for Zoonosis Control. Hokkaido University. Sapporo, Japan / Global Station for Zoonosis Control. Global Institution for Collaborative Research and Education (GI-CoRE). Hokkaido University. Sapporo, Japan / Global Virus Network. Baltimore, Maryland, USA.
Abstract
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.
Keywords
Chikungunya virus (CHIKV)
Genome
MinION sequencing
Transcription loop-mediated isothermal amplification (RT-LAMP)
 
Publisher
Public Library of Science
Citation
HAYASHIDA, Kyoko et al. Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing. PLoS Neglected Tropical Diseases, v. 13, n. 6, p. 1-15, Jun. 2019.
DOI
10.1371/journal.pntd.0007480
ISSN
1935-2727