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

21 Scopus citations


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 (US)
Article number830
JournalFrontiers in Microbiology
Issue numberAPR
StatePublished - Apr 27 2018

Bibliographical note

Funding Information:
We thank Ayumi Akiyoshi (National Institute of Polar Research, Japan) for the technical assistance. We also express our appreciation to Dr. Hiroyuki Katayama (The University of Tokyo, Japan), Dr. Kazuyoshi Yano (Tokyo Metropolitan Institute of Public Health, Japan), Prof. Osamu Nakagomi (Nagasaki University, Japan), and Dr. You Ueki (Miyagi Prefectural Institute of Public Health and Environment, Japan) for kindly providing viruses or viral cDNAs.This work was supported by JSPS KAKENHI grant numbers 15K18141, 16H04442, and 16H02371 to SK, NA, and TY; Program for the Strategic Promotion of International Cooperation to Accelerate Innovation in Developing Countries of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT); National Institute of Polar Research (NIPR) through General Collaboration Project No. 27-19; and Core Research for Evolutionary Science and Technology (CREST) from the Japan Science and Technology Agency (JST)

Publisher Copyright:
© 2018 Oshiki, Miura, Kazama, Segawa, Ishii, Hatamoto, Yamaguchi, Kubota, Iguchi, Tagawa, Okubo, Uemura, Harada, Kobayashi, Araki and Sano.


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


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