Abstract
Human noroviruses impose a considerable health burden globally. Here, a flow cytometry approach designed for their detection in biological waste and food samples was developed using antibody-coated magnetic beads. Antipeptide antibodies against murine norovirus and various human norovirus genotypes were generated for capture and coated onto magnetic beads. A flow cytometry assay was then implemented to detect bead-bound human norovirus GI.3 in patient stool samples and in norovirus-spiked mussel digestive tissues. The detection limit for stool samples was 105 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was ten-fold higher than in stool samples. Further assays showed a decrease in fluorescence intensity for heat- or UV-inactivated virus particles. Overall, we demonstrate the application of a flow cytometry approach for direct detection of small non-enveloped virus particles such as noroviruses. An adaptation of the technology to routine diagnostics has the potential to contribute a rapid and sensitive tool to norovirus outbreak investigations. Further improvements to the method, notably decreasing the detection limit of the approach, may allow the analysis of naturally contaminated food and environmental samples.
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Acknowledgements
We thank Dr Pascale Huynen (Liège CHU, Belgium), Dr Bavo Verhaegen and Dr Katelijne Dierick (Sciensano, Brussels, Belgium) for providing clinical samples. This project was supported by a Grant from the Service Public Fédéral ‘Santé Publique, Sécurité de la Chaîne alimentaire et Environnement’ (RT15/8 IQUINOR2) (R.M.R, L. F. L.-B., E. T.).
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12560_2021_9494_MOESM1_ESM.jpg
Supplementary file1 GI.3 human norovirus (HuNoV) detection in faecal suspension by flow cytometry. Gating strategy for flow cytometry analysis: forward versus side scatter (SSC vs FSC) gating (dot plots), the graphs on the right for each condition (histogram) show the fluorescence profiles. (A) Negative control. (B) GI.3 diluted 1:10, (C) 1:100, (D) 1:1000, (E) 1: 10000, (F) 1:100000, (G) 1:1000000 (JPG 10838 kb)
12560_2021_9494_MOESM2_ESM.jpg
Supplementary file2 Non-inactivated or inactivated GI.3 human norovirus (HuNoV) detection in faecal suspension by flow cytometry. Gating strategy for flow cytometry analysis: forward versus side scatter (SSC vs FSC) gating (dot plots), the graphs on the right for each condition (histogram) show the fluorescence profiles. (H) Negative control: PBS. (I) Non-inactivated GI.3. (J) Heat-inactivated GI.3. (K) UV-inactivated GI.3 (JPG 6619 kb)
12560_2021_9494_MOESM3_ESM.jpg
Supplementary file3 GI.3 human norovirus (HuNoV) detection in spiked mussel DTs by flow cytometry. Gating strategy for flow cytometry analysis: forward versus side scatter (SSC vs FSC) gating (dot plots), the graphs on the right for each condition (histogram) show the fluorescence profiles. (A) Negative control: PBS. (B) Negative control: negative mussel DTs supernatant. (C) GI.3 diluted 1:10, (D) 1:100, (E) 1:1000, (F) 1: 10000, (G) 1:100000, (H) 1:1000000 (JPG 12771 kb)
12560_2021_9494_MOESM4_ESM.jpg
Supplementary file4 Non-inactivated or inactivated GI.3 human norovirus (HuNoV) detection in spiked mussel DTs by flow cytometry. Gating strategy for flow cytometry analysis: forward versus side scatter (SSC vs FSC) gating (dot plots), the graphs on the right for each condition (histogram) show the fluorescence profiles. (I) Negative control: PBS. (J) Negative control: negative mussel DTs supernatant. (K) Non-inactivated GI.3. (L) Heat-inactivated GI.3. (M) UV-inactivated GI.3 (JPG 7899 kb)
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Razafimahefa, R.M., Ludwig-Begall, L.F., Diallo, M.A. et al. Development of a Specific Anti-capsid Antibody- and Magnetic Bead-Based Immunoassay to Detect Human Norovirus Particles in Stool Samples and Spiked Mussels via Flow Cytometry. Food Environ Virol 13, 493–506 (2021). https://doi.org/10.1007/s12560-021-09494-w
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DOI: https://doi.org/10.1007/s12560-021-09494-w