Ann Lab Med.  2023 Jan;43(1):38-44. 10.3343/alm.2023.43.1.38.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods

• Cho EJ
• Cha Y
• Lee SK
• Kim HS
• Kim JS
• Lee EJ
• Lee N
• Hong KH
• Huh HJ
• Cha YJ
• Kim HS

• KMID: 2551581
• DOI: http://doi.org/10.3343/alm.2023.43.1.38

Abstract

Background
Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials.
Methods
Norovirus GI and GII RNA sequences including the ORF1–ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements.
Results
The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at –20°C or –70°C. All data from the five laboratories were within a range of 1.0 log copies/μL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/μL and 6.96 log copies/μL, respectively.
Conclusions
The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Keyword

Noroviruses; RNA; Multicenter study; Reference material; Uncertainty; Characterization

Figure

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  Fig. 1 Overview of the development of the synthetic norovirus GI and GII RNAs.

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  Fig. 2 Long-term stability results over 1 year for the synthetic norovirus GI (A–D) and GII (E–H) reference materials. Mean Ct values at four temperatures (±SDs from two vials in triplicate) are shown. Error bars represent two SDs from six measurements. Abbreviations: NoV, norovirus; Ct, cycle threshold.

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