Broad-range and Effective Detection of Human Noroviruses by Colloidal Gold Immunochromatographic Assay Based on the Shell Domain of the Major Capsid Protein

Abstract

Background: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all ages worldwide. As the replication of HuNoVs in vitro is immature, the detection of HuNoVs is depended on molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced operators to perform, and extended time to get results. In addition, the diversity of viral genotypes is high. Therefore, a method for rapidly, broad-range, and effective detectionf of HuNoVs was desiderated for screening the excrement or vomit from infected people when outbreaks occur.Results: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for highly effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2×106 genomic copies per gram of stool sample (gc/g) and 4.4×105 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against that by RT-qPCR. The relative sensitivity, specificity and agreement of the ICA was 84.2 % (95% CI: 83.6-84.8 %), 100.0 % (95 % CI: 98.5-100.0 %) and 87.7 % (95% CI: 85.6-89.8 %), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA could detect a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs.Conclusions: Our results demonstrated that ICA targeting the S domain of VP1 is a promising candidate for effectively improve identifying different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.

The globally re-emerging norovirus GII.2 manifests higher heat resistance than norovirus GII.4 and Tulane virus

“…Two HuNoV positive stool samples were collected and genotyped as HuNoV GII.4 Sydney[P31] (NCBI accession number MZ675432) and GII.2[P16] (NCBI accession number MZ675365). After heat treatment at 60, 70, 80 and 90°C for 5 min, respectively, the heated and unheated virus suspensions were directly coated on microtiter plates and detected by monoclonal antibodies against the linear epitope of HuNoVs capsid protein with broad recognition of HuNoV genotypes (Xu et al, 2021) with ELISA. As a result, for the GII.4 Sydney[P31] sample, the detected OD 450 signals decreased drastically from 1.61 ± 0.07 to 0.60 ± 0.11 after treatment at 60°C for 5 min (37 ± 16% of the positive control, p < 0.01, Figure 3).…”

“…The treated/untreated virus suspensions were directly coated on 96‐well microtiter plates (100 μl per well) at 4°C overnight. After being washed once with PBS containing 0.05% Tween 20 (PBS‐T), the plate was blocked with 5% nonfat dried milk (Blotto, 200 μl per well) at 37°C for 1 h. The bound NoV proteins were detected with mouse monoclonal antibodies against the shell domain in the major capsid protein of HuNoVs as reported by Xu et al (2021) and by adding horseradish peroxidase‐conjugated with goat anti‐mouse IgG (1:1500; Sigma Aldrich). Horseradish peroxidase activity was detected with a TMB (3,3′,5,5′‐tetramethylbenzidine) kit (Sigma Aldrich), and the signal intensities (the optical density at 450 nm [OD 450 ]) were read with a Multiskan Sky plate reader (Thermo Fisher Scientific).…”