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

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

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.

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