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

Autor: Carolina Cardoso dos Santos, William W. Hall, Patrícia Brasil, Cintia Damasceno dos Santos Rodrigues, Guilherme Amaral Calvet, Yasuko Orba, Kyoko Hayashida, Maria Angélica Mares-Guia, Yuki Eshita, Hirofumi Sawa, Ana Maria Bispo de Filippis, Lucky Ronald Runtuwene, Yutaka Suzuki, Junya Yamagishi, Chihiro Sugimoto, Patrícia Carvalho de Sequeira
Rok vydání: 2019
Předmět:
0301 basic medicine
RNA viruses
Viral Diseases
Genotyping Techniques
Physiology
Molecular biology
RC955-962
medicine.disease_cause
Pathology and Laboratory Medicine
Genome
Biochemistry
Database and Informatics Methods
0302 clinical medicine
Arctic medicine. Tropical medicine
Medicine and Health Sciences
Chikungunya
RNA structure
Chikungunya Virus
Temperature
virus diseases
RNA alignment
3. Good health
Body Fluids
Nucleic acids
Infectious Diseases
Blood
Molecular Diagnostic Techniques
Medical Microbiology
Viral Pathogens
Viruses
Public aspects of medicine
RA1-1270
Pathogens
Anatomy
Nucleic Acid Amplification Techniques
Sequence Analysis
Brazil
Research Article
Neglected Tropical Diseases
Bioinformatics
Point-of-Care Systems
Alphaviruses
030231 tropical medicine
Loop-mediated isothermal amplification
Genomics
Biology
Research and Analysis Methods
Microbiology
DNA sequencing
Togaviruses
03 medical and health sciences
medicine
Genetics
Humans
Desiccation
Reverse Transcription Loop-mediated Isothermal Amplification
Genotyping
Microbial Pathogens
DNA sequence analysis
Gene amplification
Biology and life sciences
Public Health
Environmental and Occupational Health
Organisms
Chikungunya Infection
Reverse Transcription
Sequence Analysis
DNA
Tropical Diseases
Virology
Macromolecular structure analysis
030104 developmental biology
RNA amplification
Minion
Chikungunya Fever
RNA
Sequence Alignment
Zdroj: PLoS Neglected Tropical Diseases
PLoS Neglected Tropical Diseases, Vol 13, Iss 6, p e0007480 (2019)
ISSN: 1935-2735
Popis: 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.
Databáze: OpenAIRE
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