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

doi:10.3389/fmicb.2018.00830

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

Front Microbiol. 2018 Apr 27:9:830. doi: 10.3389/fmicb.2018.00830. eCollection 2018.

Authors

Affiliations

¹ Department of Civil Engineering, National Institute of Technology, Nagaoka, Japan.
² Department of Environmental Health, National Institute of Public Health, Wako, Japan.
³ Center for Simulation Sciences and Informational Biology, Ochanomizu University, Bunkyô, Japan.
⁴ Center for Life Science Research, University of Yamanashi, Kofu, Japan.
⁵ Department of Soil, Water and Climate, University of Minnesota, Minneapolis, MN, United States.
⁶ Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Japan.
⁷ Department of Science of Technology Innovation, Nagaoka University of Technology, Nagaoka, Japan.
⁸ Department of Civil and Environmental Engineering, Tohoku University, Sendai, Japan.
⁹ Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan.
¹⁰ New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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.

Keywords: MiSeq sequencing; high-throughput detection and genotyping; human feces; human pathogenic viruses; microfluidic PCR; oyster; sewage.