Shanghai Entry-Exit Inspection and Quarantine Bureau, Shanghai 200135, China.
Food Microbiol. 2012 Jun;30(2):420-6. doi: 10.1016/j.fm.2011.12.026. Epub 2012 Jan 11.
In this study, we developed a sensitive receptor-binding capture and magnetic sequestration (RBCMS) method capable of concentrating human norovirus (HuNoV) from various food samples within few hours. We found that distilled water was suitable for the elution of HuNoV from inoculated tomatoes and blueberries, and glycine buffer improved the elution of HuNoV from inoculated salad. A significant improvement in post-extraction RNA yield was achieved by sequentially heat-releasing and column-extracting over either technique alone. The viral recovery of the RBCMS method was significantly higher than both the same-day PEG method (90 min PEG precipitation) and the two-day PEG method (overnight PEG precipitation) with a recovery rate of 8.75%, 1.03% and 5.40%, respectively. The detection limit of HuNoV by RBCMS method was significantly improved to 0.056 RTU. The estimated minimal concentration powers (MCPs) were 6.11, 30.48, and 63.60-fold for the same-day PEG, two-day PEG, and RBCMS methods, respectively. RNase protection assay suggests that the viral genome was protected from RNase attack by remaining within the viral capsid. The signal detected by the RBCMS method might be more biologically relevant, as it requires both intact viral capsid to bind to HBGA receptors and the presence of viral genome to be amplified. Overall, the RBCMS method takes significantly less time than current PEG precipitation methods, recovers a higher yield of HuNoV from various food samples, and hence exhibits higher sensitivity.
在本研究中，我们开发了一种灵敏的受体结合捕获和磁性分离（RBCMS）方法，能够在数小时内从各种食物样本中浓缩人诺如病毒（HuNoV）。我们发现蒸馏水适合洗脱接种番茄和蓝莓中的 HuNoV，甘氨酸缓冲液可提高接种沙拉中 HuNoV 的洗脱效率。与单独使用任何一种技术相比，顺序热释放和柱提取均可显著提高提取后 RNA 的产量。RBCMS 方法的病毒回收率明显高于同一天的 PEG 方法（90 分钟 PEG 沉淀）和两天的 PEG 方法（过夜 PEG 沉淀），回收率分别为 8.75%、1.03%和 5.40%。RBCMS 方法检测 HuNoV 的检测限显著提高至 0.056 RTU。RBCMS 方法的估计最小浓度能力（MCP）分别为同一天 PEG、两天 PEG 和 RBCMS 方法的 6.11、30.48 和 63.60 倍。RNase 保护分析表明，病毒基因组通过保留在病毒衣壳内而免受 RNase 攻击。RBCMS 方法检测到的信号可能更具生物学相关性，因为它需要完整的病毒衣壳与 HBGA 受体结合，并且存在病毒基因组才能进行扩增。总的来说，RBCMS 方法比目前的 PEG 沉淀方法耗时更短，从各种食物样本中回收更高产量的 HuNoV，因此具有更高的灵敏度。
