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

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 10 5 gc/mL, thus bettering detection limits of commercially available norovirus diagnosis quick kits of 100-fold; the detection limit in spiked mussels however was tenfold 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.

Recent Advances in Biosensor Development for the Detection of Viral Particles in Foods: A Comprehensive Review

“…The recovery rates for HAV were 3−21-fold higher than those obtained when using the ISO 15216-1:2017 method. Razafimahefa et al 42 developed an immunomagnetic separation-based method for the detection of NoV in mussels: magnetic beads conjugated with anti-NoV peptide antibodies were used to capture NoV in a mussel homogenate. The immunomagnetic separation method combined with flow cytometry assays has a detection limit 10-fold lower than that of commercial kits.…”

Preparation of monoclonal antibodies against norovirus and establishment of a rapid immunochromatographic technique

“…At present, the main diagnostic methods for NoV include electron microscopy, 13 molecular biology assays 14,15 and immunoassay. 16 Conventional RT-PCR is recognized as the ''gold standard'' for NoV detection, because of its advantages of simple operation, high sensitivity and wide application range. 17,18 For example, Miura et al 19 established a one-step real-time fluorescence quantitative RT-PCR method, which includes three pairs of primers targeting the three gene groups of NoV (GI, GII, and GIV), with strong specificity and high sensitivity.…”