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From January to December 2016, a total of 480 oyster samples were collected in Guangxi. We obtained 240 samples from the two largest farms situated along the coastline in Guangxi, and a further 120 samples were purchased from wholesalers and retailers at supermarkets in three major cities in Guangxi. The remaining 120 samples were collected from restaurants where seafood was served (Table 1).
Sampling Site No. Sampled No. Acceptable Results Mean Extraction Efficiency (%) No. Positive (%) GⅠ GⅡ Total Farms 240 232 8.6 ± 2.3 1 47 48 (20.7) Markets 120 116 9.8 ± 3.4 1 27 28 (24.1) Restaurants 120 115 7.9 ± 2.8 0 20 20 (17.4) All 480 463 8.8 ± 2.9 2 (0.4) 94 (20.3) 96 (20.7) Table 1. Extraction Efficiencies and Norovirus Detection in Oysters by Sampling Site
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Of 480 samples tested, 463 (96.5%) provided valid results (i.e., with > 1% extraction efficiency and > 10, 000 event numbers) and were used for further analysis. Four samples with unacceptable extraction efficiency and 13 samples showing low number of total droplets were consequently excluded from the study. Extraction efficiency values ranged from 3.5%-13.2% (mean = 8.8% ± 2.9%), with no difference between samples from farms, markets, or restaurants (Table 1).
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Of the 463 samples analyzed, 96 (20.7%) tested positive for norovirus, with 94 (20.3%) being positive for GⅡ and 2 (0.4%) being positive for GⅠ. Figure 1 shows the visual representation of six oyster samples that were positive for norovirus (event numbers > 10, 000), as indicated by RT-ddPCR. The prevalence varied between sampling locations, but a comparison of the norovirus prevalence using Bonferroni correction revealed no significant difference in the site-by-site prevalence (α = 0.05, α' = 0.0125) (Table 1). Norovirus was detected throughout the year, but the prevalence showed an obvious seasonal difference (P < 0.001) between the summer months (11.0%) and the winter months (30.2%).
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Quantitative data showed that levels of norovirus in the tested samples varied widely (up to 100-fold). The highest level of norovirus was 314, 300 copies/g digestive gland, while the lowest level was 6, 894 copies/g. The geometric mean of total norovirus (GⅠ and GⅡ) was 19, 300 copies/g; 22, 520 copies/g for GⅠ, and 19, 230 copies/g for GⅡ.
Among the oyster samples quantified, 54.2% (52/96) ranged from 10, 000 to 100, 000 copies/g, and 41.7% (40/96) were below 10, 000 copies/g. Four (4.2%) samples containing over 100, 000 copies/g were detected between December and February, when the highest rates of norovirus positivity were recorded. Norovirus levels also varied between sampling sites, with the highest geometric mean score observed in the samples collected from farms and the lowest in the samples from restaurants (Table 2).
Sampling Site No. Quantified No. Positive within Quantity Ranges (copies/g) Geometric Mean
(copies/g)> 100, 000 10, 000-100, 000 < 10, 000 Farms 232 1 26 21 21, 350 Markets 116 3 14 11 18, 590 Restaurants 115 0 12 8 15, 400 All 463 4 52 40 19, 300 Table 2. Norovirus Levels in Oysters by Sampling Site
Norovirus levels in positive samples showed marked seasonal variation (Figure 2). Norovirus concentrations were significantly greater from November to April than from May to October. For each month of the study, the percentage of samples with total norovirus in different quantity ranges is shown in Figure 3. Both the prevalence and levels of norovirus showed strong seasonality (highest levels during winter months and lowest levels during summer months).
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As shown in Table 3, the sensitivity of norovirus detection by real-time RT-PCR was approximately 1.88 × 102 copies/µL, while RT-ddPCR quantification was applicable for norovirus with ≥ 1.88 × 101 copies/µL. Thus, RT-ddPCR showed a ten-fold greater sensitivity than real-time RT-PCR for detection of norovirus. However, RT-ddPCR reported 9 ± 7, 105 ± 56, and 1, 220 ± 296 copies/µL in the 1.88 × 101, 1.88 × 102, and 1.88 × 103 copies/µL norovirus standards, respectively, counting approximately 40%-50% fewer RNA copies than expected relative to the input standard copy number.
Norovirus Standards
(copies/µL)Real-time RT-PCR RT-ddPCR (copies/µL) Result Wells detected/tested Result Wells detected/tested 1.88 × 100 - 0/5 - 0/5 1.88 × 101 - 0/5 9 ± 7 4/5 1.88 × 102 + 5/5 105 ± 56 5/5 1.88 × 103 + 5/5 1, 120 ± 296 5/5 Table 3. Comparison of Real-time RT-PCR and RT-PCR Measurements of Norovirus Plasmid Standards with Different Viral Loads