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 plasmidDNAcontaining the target sequences, it was confirmed that both assays allow detection and quantification of AiVs with a quantitative range of 1.0×101 to 1.0×107 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 AiVRNA loads were determined to be 1.4×104 to 6.6×109 copies/g stool.Wealso 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 RNAconcentrations in influent and effluent wastewater were determined to be up to 2.2×107 and 1.8×104 copies/liter, respectively. Our RT-qPCR system is useful for routine diagnosis of AiVs in clinical stool specimens and environmental samples. © 2013, American Society for Microbiology.