Division of Molecular Biology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Road, Laurel, Maryland 20708, USA.
J Food Prot. 2018 Jan;81(1):105-114. doi: 10.4315/0362-028X.JFP-17-134.
The development of rapid and sensitive detection methods for human noroviruses (HuNoV) in produce items is critical, especially with the recent rise in outbreaks associated with this food commodity. In this study, 50-g portions of various produce items linked to a norovirus outbreak (celery, cucumber, lettuce, grapes, and radish) were artificially inoculated with murine norovirus (MNV-1) and concentrated either by ultracentrifugation or polyethylene glycol (PEG) precipitation after elution with an alkaline Tris-glycine-beef extract buffer supplemented with pectinase. As a viral concentration step following virus elution and clarification, ultracentrifugation yielded a faster method (<8 h, including reverse transcription quantitative PCR), with MNV-1 recoveries similar to or better, than those obtained with PEG precipitation. The addition of polyvinylpyrrolidone to the elution buffer, to remove polyphenolic inhibitors, improved MNV-1 recoveries by over two- and fivefold for cucumber and grapes, respectively. However, despite MNV-1 recoveries ranging from 10 to 38% as calculated with 10-fold diluted RNA, contaminating HuNoV was not detected in any of the outbreak-associated samples tested. For store-bought produce samples, the limit of detection for artificially seeded HuNoV GII.4 was determined to be 10 copies per 50 g, with reproducible detection achieved in grapes, radish, and celery. The results support the use of ultracentrifugation as an alternative approach to PEG precipitation to concentrate norovirus from a variety of produce items.
开发快速、灵敏的检测方法对于人类诺如病毒（HuNoV）在农产品中的检测至关重要，尤其是在与这种食品相关的疫情近期不断上升的情况下。在本研究中，将 50 克与诺如病毒疫情相关的各种农产品（芹菜、黄瓜、生菜、葡萄和萝卜）部分进行人工接种鼠诺如病毒（MNV-1），然后用碱性三羟甲基氨基甲烷-甘氨酸-牛提取物缓冲液（添加果胶酶）洗脱后，通过超速离心或聚乙二醇（PEG）沉淀浓缩。作为病毒洗脱和澄清后的病毒浓缩步骤，超速离心法（<8 小时，包括逆转录定量 PCR）速度更快，MNV-1 的回收率与 PEG 沉淀法相当或更好。在洗脱缓冲液中添加聚乙烯吡咯烷酮以去除多酚抑制剂，可分别使黄瓜和葡萄中的 MNV-1 回收率提高两倍和五倍以上。然而，尽管根据 10 倍稀释 RNA 计算，MNV-1 的回收率在 10%到 38%之间，但在测试的任何与疫情相关的样本中均未检测到污染的人类诺如病毒。对于商店购买的农产品样本，人工接种的人类诺如病毒 GII.4 的检测限确定为每 50 克 10 个拷贝，在葡萄、萝卜和芹菜中可实现重复性检测。结果支持使用超速离心代替 PEG 沉淀法来浓缩各种农产品中的诺如病毒。
