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

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.