Microfluidic PCR amplification and MiSeq amplicon sequencing techniques for high-throughput detection and genotyping of human pathogenic RNA viruses in human feces, sewage, and oysters

Mamoru Oshiki, Takayuki Miura, Shinobu Kazama, Takahiro Segawa, Satoshi Ishii, Masashi Hatamoto, Takashi Yamaguchi, Kengo Kubota, Akinori Iguchi, Tadashi Tagawa, Tsutomu Okubo, Shigeki Uemura, Hideki Harada, Naohiro Kobayashi, Nobuo Araki, Daisuke Sano

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI), GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101-104 copies/mL-sewage, 105-108 copies/g-human feces, and 102-105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

Original languageEnglish
Article number830
JournalFrontiers in Microbiology
Volume9
Issue numberAPR
DOIs
Publication statusPublished - 2018 Apr 27

Keywords

  • High-throughput detection and genotyping
  • Human feces
  • Human pathogenic viruses
  • MiSeq sequencing
  • Microfluidic PCR
  • Oyster
  • Sewage

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

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