Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.
Kyoko HayashidaYasuko OrbaPatricia C SequeiraChihiro SugimotoWilliam W HallYuki EshitaYutaka SuzukiLucky RuntuwenePatricia BrasilGuilherme CalvetCintia D S RodriguesCarolina C Dos SantosMaria A M Mares-GuiaJyunya YamagishiAna M B de FilippisHirofumi SawaPublished in: PLoS neglected tropical diseases (2019)
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.
Keyphrases
- loop mediated isothermal amplification
- sensitive detection
- single cell
- genome wide
- high throughput
- zika virus
- end stage renal disease
- ejection fraction
- randomized controlled trial
- transcription factor
- newly diagnosed
- gene expression
- chronic kidney disease
- dna methylation
- machine learning
- quantum dots
- genetic diversity
- molecularly imprinted
- patient reported outcomes
- nucleic acid
- data analysis