Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing / 生物医学与环境科学(英文)
Biomedical and Environmental Sciences
;
(12): 41-46, 2016.
Artigo
em Inglês
| WPRIM
| ID: wpr-258854
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform.</p><p><b>METHODS</b>Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance.</p><p><b>RESULTS</b>The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus.</p><p><b>CONCLUSION</b>The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Vírus da Influenza A
/
Variação Genética
/
RNA Viral
/
Genoma Viral
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Genética
/
Métodos
Tipo de estudo:
Guia de Prática Clínica
Idioma:
Inglês
Revista:
Biomedical and Environmental Sciences
Ano de publicação:
2016
Tipo de documento:
Artigo
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