European Union Reference Laboratory (EURL) for Foodborne Viruses, National Food Agency, Hamnesplanaden 5, 753 19 Uppsala, Sweden; Department of Medical Sciences, Zoonosis Science Centre, Uppsala University, Husargatan 3, 752 37 Uppsala, Sweden.
European Union Reference Laboratory (EURL) for Foodborne Viruses, National Food Agency, Hamnesplanaden 5, 753 19 Uppsala, Sweden.
Int J Food Microbiol. 2020 Feb 16;315:108386. doi: 10.1016/j.ijfoodmicro.2019.108386. Epub 2019 Nov 7.
The NucliSENS MiniMAG (Minimag) system from bioMérieux is widely used for extraction of viral RNA from oysters and is included as informative material in the ISO method for quantification of hepatitis A virus (HAV) and norovirus genogroups I and II (GI and GII) in food (ISO 15216-1:2017). However, the system is no longer on sale within the EU and alternative methods are therefore needed. We optimised and evaluated an automated benchtop system for extraction of viral RNA from oysters artificially contaminated with HAV, norovirus GI, norovirus GII and mengovirus, using the same reagents and a similar protocol as with the Minimag method. Using the automated system instead of Minimag increased measured viral concentration by on average 1.3 times, suggesting that the automated system extracts viral RNA more efficiently than Minimag. A drawback with the automated system was that it displayed higher variability in measured concentration for mengovirus. The median viral recovery was 17%, 37%, 44% and 41% for samples extracted with the automated system and 15%, 27%, 34% and 23% for samples extracted with Minimag for HAV, norovirus GI, norovirus GII and mengovirus, respectively. All samples displayed <75% inhibition in RT-qPCR when extracted with the automated system or Minimag. Together, these results suggest that the automated system can be a suitable alternative to Minimag in analysis of HAV, norovirus GI and norovirus GII in oysters. However, verification using naturally contaminated oysters is needed before it can be used for food safety control purposes.
生物梅里埃的 NucliSENS MiniMAG（Minimag）系统广泛用于从牡蛎中提取病毒 RNA，并且已包含在 ISO 方法中，用于定量食品中的甲型肝炎病毒（HAV）和诺如病毒基因组 I 和 II 型（GI 和 GII）（ISO 15216-1:2017）。然而，该系统已不在欧盟范围内销售，因此需要替代方法。我们优化并评估了一种自动化台式系统，用于从人工污染 HAV、诺如病毒 GI、诺如病毒 GII 和肠道病毒的牡蛎中提取病毒 RNA，该系统使用与 Minimag 方法相同的试剂和类似的方案。与 Minimag 相比，使用自动化系统可使测量的病毒浓度平均增加 1.3 倍，这表明自动化系统比 Minimag 更有效地提取病毒 RNA。自动化系统的一个缺点是，它对肠道病毒的测量浓度显示出更高的变异性。对于使用自动化系统提取的样本，HAV、诺如病毒 GI、诺如病毒 GII 和肠道病毒的病毒回收率中位数分别为 17%、37%、44%和 41%，而对于使用 Minimag 提取的样本，病毒回收率中位数分别为 15%、27%、34%和 23%。当使用自动化系统或 Minimag 提取时，所有样本的 RT-qPCR 抑制率均<75%。总的来说，这些结果表明，在分析牡蛎中的 HAV、诺如病毒 GI 和诺如病毒 GII 时，自动化系统可以替代 Minimag。然而，在将其用于食品安全控制目的之前，需要使用受自然污染的牡蛎进行验证。
