Dataset Information

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

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.

SUBMITTER: Cho EJ

PROVIDER: S-EPMC9467847 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Cho Eun-Jung EJ Cha Younggil Y Lee Su Kyung SK Kim Han-Sung HS Kim Jae-Seok JS Lee Eun Jin EJ Lee Nuri N Hong Ki Ho KH Huh Hee Jin HJ Cha Young Joo YJ Kim Hyun Soo HS

Annals of laboratory medicine 20220901 1

<h4>Background</h4>Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials.<h4>Methods</h4>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 <i>in vitro</i> transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial nor ...[more]

PMID: 36045055

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Project description:BackgroundQuantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods.ResultsDNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/μL. Forty-seven extracts had concentrations greater than 17.5 ng/μL, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ μL), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/μL to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/μL and 0.25 ng/μL respectively. The qPCR LOQ varied by cell type (5.72 × 10-3 ng/μL for E. coli, 2.66 × 10-3 ng/μL, for B. cereus, 3.78 × 10-3 ng/μL for B. thailandensis, and 7.67 × 10-4 ng/μL for S. cerevisiae). A number of samples were below the UV spectroscopy (n = 27), flurometry (n = 15), and qPCR (n = 3) LOQ.ConclusionThe presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.

Dataset Information

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

Background

Methods

Results

Conclusions

Publications

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

Similar Datasets

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