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

Frontiers in Microbiology
Mamoru OshikiDaisuke Sano

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....Continue Reading

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Aug 26, 2019·Food and Environmental Virology·Sofia StrubbiaFrançoise S Le Guyader
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Apr 21, 2021·Food Microbiology·Bruna Leal MaskeCarlos Ricardo Soccol

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Datasets Mentioned

BETA
DRA005567

Methods Mentioned

BETA
genotyping
PCR
chip
PCRs
amplicon
amplicon sequencing
Assay
electrophoresis

Software Mentioned

ClustalW
MacQIIME
usearch
NoV Genotyping Tool
MEGA7
blastn

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