Development of a Reverse Transcription-Quantitative PCR System for Detection and Genotyping of Aichi Viruses in Clinical and Environmental Samples

Abstract

dAichi 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.

Temporal variations in genotype distribution of human sapoviruses and Aichi virus 1 in wastewater in Southern Arizona, United States

“… AB, AiV‐1 universal assay; A, genotype A‐specific assay; B, genotype B‐specific assay (Kitajima et al . ). …”

Pepper mild mottle virus as an indicator and a tracer of fecal pollution in water environments: Comparative evaluation with wastewater-tracer pharmaceuticals in Hanoi, Vietnam

“…The methods used to extract the viral genomes and to quantify PMMoV and HEV by quantitative PCR (qPCR) was based on those in the previous studies Katayama et al, 2008;Kitajima et al, 2013;Zhang et al, 2006). Briefly, for RNA viruses (PMMoV, AiV1, NoV GI, NoV GII and EV), the viral RNA was extracted from 140 μL of each further-concentrated sample by using a QIAamp Viral RNA Minikit (Qiagen, the Netherlands) followed by reverse transcription (RT) for cDNA synthesis with a High Capacity cDNA Reverse Transcription Kit (Life Technologies, CA).…”

Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

“…Previously validated TaqMan qPCR assays (10)(11)(12)(13)(30)(31)(32)(33)(34)(35)(36) were used for this study (Table 1). These assays have been successfully applied to specifically quantify viruses in water and other environmental samples.…”