Journal
FRONTIERS IN MICROBIOLOGY
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fmicb.2018.00830
Keywords
human pathogenic viruses; high-throughput detection and genotyping; microfluidic PCR; MiSeq sequencing; sewage; human feces; oyster
Categories
Funding
- JSPS KAKENHI [15K18141, 16H04442, 16H02371]
- Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT)
- National Institute of Polar Research (NIPR) [27-19]
- Core Research for Evolutionary Science and Technology (CREST) from the Japan Science and Technology Agency (JST)
- Grants-in-Aid for Scientific Research [15K18141, 16H02371, 16H04442, 15H05339] Funding Source: KAKEN
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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 10 degrees to 10(3) copies/mu L in cDNA sample, corresponding to 10(1)-10(4) copies/mL-sewage, 10(5)-10(5) copies/g-human feces, and 10(2)-10(5) 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.
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