• The RT-dPCR method was established to accurately quantify the pseudovirus RM for norovirus GⅡ.4.
• The pseudovirus RM for norovirus GⅡ.4 comprises ORF2 RNA and demonstrates homogeneity and stability.
• The pseudovirus RM with an assigned value can serve as a quality control tool for the detection methodology targeting norovirus GⅡ.4.
The human norovirus (NV) is widely recognized as a pathogen causing acute gastroenteritis, particularly associated with elevated morbidity and mortality rates in pediatric and geriatric populations. While current detection methods, such as those based on immune reactions and quantitative polymerase chain reaction (qPCR), have been developed by researchers, these techniques do not provide absolute quantification. In this study, we established a reference material (RM) comprising pseudovirus particles containing NV ORF2 RNA. The RM was quantified using reverse transcription digital PCR (RT-dPCR), demonstrating ideal homogeneity and stability. The assigned value of the RM, including expanded uncertainty, was determined to be (3.42 ± 0.69) × 103 copies/μL. This RM can be effectively utilized to validate commercial diagnostic kits and platforms, thereby enhancing the accuracy and reliability of acute gastroenteritis diagnosis.
