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Development of a Reverse Transcription-Quantitative PCR System for Detection and Genotyping of Aichi Viruses in Clinical and Environmental Samples
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- Masaaki Kitajima
- Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, Arizona, USA
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- Akihiko Hata
- Department of Urban Engineering, The University of Tokyo, Tokyo, Japan
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- Teruo Yamashita
- Department of Microbiology and Medical Zoology, Aichi Prefectural Institute of Public Health, Nagoya, Aichi, Japan
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- Hiroyuki Katayama
- Department of Urban Engineering, The University of Tokyo, Tokyo, Japan
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- Eiji Haramoto
- International Research Center for River Basin Environment, University of Yamanashi, Kofu, Yamanashi, Japan
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- Hiroko Minagawa
- Department of Microbiology and Medical Zoology, Aichi Prefectural Institute of Public Health, Nagoya, Aichi, Japan
Bibliographic Information
- Published
- 2013-07
- Resource Type
- journal article
- Rights Information
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- https://journals.asm.org/non-commercial-tdm-license
- DOI
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- 10.1128/aem.00820-13
- Publisher
- American Society for Microbiology
Search this article
Description
<jats:title>ABSTRACT</jats:title> <jats:p> Aichi viruses (AiVs) have been proposed as a causative agent of human gastroenteritis potentially transmitted by fecal-oral routes through contaminated food or water. In the present study, we developed a TaqMan minor groove binder (MGB)-based reverse transcription-quantitative PCR (RT-qPCR) system that is able to quantify AiVs and differentiate between genotypes A and B. This system consists of two assays, an AiV universal assay utilizing a universal primer pair and a universal probe and a duplex genotype-specific assay utilizing the same primer pair and two genotype-specific probes. The primers and probes were designed based on multiple alignments of the 21 available AiV genome sequences containing the capsid gene. Using a 10-fold dilution of plasmid DNA containing the target sequences, it was confirmed that both assays allow detection and quantification of AiVs with a quantitative range of 1.0 × 10 <jats:sup>1</jats:sup> to 1.0 × 10 <jats:sup>7</jats:sup> copies/reaction, and the genotype-specific assay reacts specifically to each genotype. To validate the newly developed assays, 30 clinical stool specimens were subsequently examined with the assays, and the AiV RNA loads were determined to be 1.4 × 10 <jats:sup>4</jats:sup> to 6.6 × 10 <jats:sup>9</jats:sup> copies/g stool. We also examined 12 influent and 12 effluent wastewater samples collected monthly for a 1-year period to validate the applicability of the assays for detection of AiVs in environmental samples. The AiV RNA concentrations in influent and effluent wastewater were determined to be up to 2.2 × 10 <jats:sup>7</jats:sup> and 1.8 × 10 <jats:sup>4</jats:sup> copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples. </jats:p>
Journal
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- Applied and Environmental Microbiology
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Applied and Environmental Microbiology 79 (13), 3952-3958, 2013-07
American Society for Microbiology
