Development of RNA reference materials for norovirus GI and GII using digital PCR.

Norovirus is a highly virulent pathogen that causes enteritis in all age groups worldwide. Owing to the diversity of noroviruses, the development of vaccines and treatments is challenging, and an early and accurate diagnosis is crucial. Reference materials (RMs) developed previously for norovirus genotypes I (GI) and II (GII) were quantified using reverse transcription quantitative PCR. In this study, we developed norovirus GI and GII RMs as in vitro transcribed RNA forms. These RMs were then assigned reference values for the RNA copy number concentration. The concentrations of GI and GII RMs determined using in-house reverse transcription digital PCR assays were (1.92±0.37)×10⁷ and (1.20±0.27)×10⁷ copy/mL, respectively. The homogeneity and stability of the RMs were evaluated, and their compatibility with commercial diagnostic kits was validated. These RMs can be used for the development of detection assays, as calibrants for various molecular measurement techniques, and as test materials for internal and external quality assurance.