Detection and Quantification of Noroviruses in Shellfish
Detection and Quantification of Noroviruses in Shellfish
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DOI：
10.1128/aem.01507-08
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发表时间：
2009-02-01
影响因子：
4.4
通讯作者：
Atmar, Robert L.
中科院分区：
文献类型：
--
作者：
Le Guyader, Francoise S.;Parnaudeau, Sylvain;Atmar, Robert L.
诺如病毒（NoV）是人类急性胃肠炎最常见的病毒因子，并且高浓度的NoV被排放到环境中。由于这些病毒很难灭活，卫生后果是污染食物，包括软体动物贝类。贝类样品中的NoV检测存在四个主要问题：低水平的病毒污染，有效的病毒提取困难，存在抑制分子检测的干扰物质，以及NoV遗传变异性。本研究的目的是调整试剂盒，使其与之前证明有效检测贝类中NoV的方法一起使用，并使用一步实时逆转录PCR方法并添加外部病毒对照。使用生物积累的牡蛎的两种方法的比较表明，该方法可重复检测牡蛎样品中的病毒水平相似。使用自然污染样品进行的验证研究也表明，两种方法的结果之间存在良好的相关性，变异性更多地归因于样品污染水平。磁性二氧化硅非常有效地消除了抑制剂，并且提取和扩增控制的使用提高了质量保证。这些控制措施增加了贝类样品中NoV浓度估计值的置信度，并有力地支持了以下结论，即本文所述方法的结果反映了牡蛎中的病毒污染水平。这种方法对食品安全很重要，目前正在评估欧洲法规。
Noroviruses (NoVs) are the most common viral agents of acute gastroenteritis in humans, and high concentrations of NoVs are discharged into the environment. As these viruses are very resistant to inactivation, the sanitary consequences are contamination of food, including molluscan shellfish. There are four major problems with NoV detection in shellfish samples: low levels of virus contamination, the difficulty of efficient virus extraction, the presence of interfering substances that inhibit molecular detection, and NoV genetic variability. The aims of this study were to adapt a kit for use with a method previously shown to be efficient for detection of NoV in shellfish and to use a one step real-time reverse transcription-PCR method with addition of an external viral control. Comparisons of the two methods using bioaccumulated oysters showed that the methods reproducibly detected similar levels of virus in oyster samples. Validation studies using naturally contaminated samples also showed that there was a good correlation between the results of the two methods, and the variability was more attributable to the level of sample contamination. Magnetic silica very efficiently eliminated inhibitors, and use of extraction and amplification controls increased quality assurance. These controls increased the confidence in estimates of NoV concentrations in shellfish samples and strongly supported the conclusion that the results of the method described here reflected the levels of virus contamination in oysters. This approach is important for food safety and is under evaluationfor European regulation.