RESUMO
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.
Assuntos
Febre de Chikungunya/diagnóstico , Vírus Chikungunya/isolamento & purificação , Técnicas de Genotipagem/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Análise de Sequência de DNA/métodos , Brasil , Febre de Chikungunya/virologia , Vírus Chikungunya/classificação , Vírus Chikungunya/genética , Dessecação , Humanos , Transcrição Reversa , TemperaturaRESUMO
Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan. We diagnosed them as CL non-invasively by the direct boil loop-mediated isothermal amplification method and an innovative sequencing method using the MinION™ sequencer. This report demonstrates that our procedure could be useful for the diagnosis of CL in both clinical and epidemiological settings.