期刊
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
卷 79, 期 13, 页码 3952-3958出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.00820-13
关键词
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资金
- Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (JST)
- Grants-in-Aid for Scientific Research [23590166] Funding Source: KAKEN
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 x 10(1) to 1.0 x 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 x 10(4) to 6.6 x 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 x 10(7) and 1.8 x 10(4) copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples.
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