title: Development of a reverse transcription-quantitative PCR system for detection and genotyping of aichi viruses in clinical and environmental samples.
abstract: 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(1) to 1.0 × 10(7) 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(4) to 6.6 × 10(9) 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(7) and 1.8 × 10(4) copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples.
authors.affiliation: Department of Soil, Water and Environmental Science, The University of Arizona, Tucson, AZ, USA. kitajima@email.arizona.edu
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kitajima
authors.forename: Akihiko
authors.initials: A
authors.lastname: Hata
authors.forename: Teruo
authors.initials: T
authors.lastname: Yamashita
authors.forename: Eiji
authors.initials: E
authors.lastname: Haramoto
authors.forename: Hiroko
authors.initials: H
authors.lastname: Minagawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Katayama
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes; D005243:Feces / Q000821:virology*; D005759:Gastroenteritis / Q000821:virology*; D005838:Genotype; D006801:Humans; D007564:Japan; D052699:Kobuvirus / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D013045:Species Specificity; D062065:Wastewater / Q000821:virology*; D014871:Water Microbiology
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D062065:Wastewater