Reverse transcription-quantitative PCR assays for genotype-specific detection of human noroviruses in clinical and environmental samples

Abstract

Circulation of human noroviruses in water environments is suspected to be genotype-dependent, but the established primer and probe sets for noroviruses are usually genogroup-specific, which do not allow to compare the genotype-specific properties, such as persistence in water environments and resistance to disinfectants. In this study, quantitative PCR assays were designed for genotype-specific quantification of four epidemiologically important genotypes, GII.3, GII.4, GII.6, and GII.17. Developed assays were tested using norovirus positive stool samples which were previously confirmed to present target genotypes of this study. The results were 100% in accordance with the previous results. Effect of the co-existence of multiple genotypes in a sample on the target genotype quantification was evaluated using composite stool samples and wastewater samples containing multiple genotypes and the presence of non-target genotypes didn't affect the quantification of target genotype. Sensitivity and specificity was 100% for all four assays developed in this study with no cross-reactions between genotypes demonstrating the validity of our assays and their applicability to clinical and environmental samples.

Keywords: Genotype; Monitoring; Norovirus; Primers; Quantification; RT-qPCR.