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

Area of Science:

• Virology
• Molecular Biology
• Biotechnology

Background:

• Norovirus is a significant global cause of enteritis across all age groups.
• The genetic diversity of noroviruses complicates vaccine development and treatment strategies.
• Accurate and early diagnosis of norovirus infections is essential.

Purpose of the Study:

• To develop and characterize novel reference materials (RMs) for norovirus genotypes I (GI) and GII.
• To establish precise RNA copy number concentrations for these RMs.
• To validate the utility of these RMs in molecular diagnostic applications.

Main Methods:

• Development of norovirus GI and GII RMs using in vitro transcription of RNA.
• Quantification of RMs using in-house reverse transcription digital PCR (RT-dPCR).
• Assessment of RM homogeneity, stability, and compatibility with commercial diagnostic kits.

Main Results:

• Norovirus GI and GII RMs were successfully produced as in vitro transcribed RNA.
• Reference values for RNA copy number concentration were assigned: (1.92±0.37)×10⁷ copy/mL for GI and (1.20±0.27)×10⁷ copy/mL for GII.
• The RMs demonstrated satisfactory homogeneity, stability, and compatibility with existing diagnostic assays.

Conclusions:

• The developed norovirus GI and GII RNA RMs serve as valuable tools for molecular diagnostics.
• These RMs can be utilized as calibrants and for quality assurance in norovirus detection assays.
• The availability of these RMs will aid in the development and standardization of norovirus detection methods worldwide.