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.
Publication types
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Evaluation Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Brazil
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Chikungunya Fever / diagnosis*
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Chikungunya Fever / virology
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Chikungunya virus / classification
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Chikungunya virus / genetics
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Chikungunya virus / isolation & purification*
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Desiccation
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Genotyping Techniques / methods*
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Humans
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Molecular Diagnostic Techniques / methods*
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Nucleic Acid Amplification Techniques / methods*
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Point-of-Care Systems*
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Reverse Transcription
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Sequence Analysis, DNA / methods*
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Temperature
Grants and funding
This study was funded by AMED program of the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID; JP18fm0108008) and Research Program on Emerging and Re-emerging Infectious Diseases (JP18fk0108108) and Japan Society for the Promotion of Science (JSPS) KAKENHI (16H05805). This study was also supported by Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H06429, 16H06431, 16K21723) and OSIMO foundation. The Flavivirus Laboratory was funded by Faperj (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro) under the grant no. E-26/2002.930/2016, by European Union’s Horizon 2020 program under grant agreements ZIKACTION no. 734857 and ZIKAPLAN no. 734548. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.