A field diagnosis and genotyping of chikungunya virus by a dried RT-LAMP and MinION sequencing

Detection and sequencing of chikungunya virus (CHIKV) genome was performed using modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and MinION sequencer. We developed the protocol for drying all the reagents for RT-LAMP in a single reaction tube with trehalose. The dried RT-LAMP tube is stable at room temperature, therefore it is not necessary to use cold-chain for providing the system to the remote area where chikungunya is endemic. Using the established system, CHIKV genome was effectively amplified under isothermal condition, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were validated with RNAs and serum samples from chikungunya patients in Brazil. The obtained sequence data was informative to recognize recent CHIKV outbreak genotype.