Quang Le H, Suffredini E, Tien Pham D, Kim To A, De Medici D
School of Biotechnology and Food Technology, Hanoi University of Science and Technology, Hanoi, Vietnam.
Department of Food Safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Rome, Italy.
Lett Appl Microbiol. 2018 Nov;67(5):426-434. doi: 10.1111/lam.13065. Epub 2018 Oct 4.
The detection of foodborne viruses in bivalve molluscs is a challenging procedure in relation to low virus concentration and to the presence of significant RT-PCR inhibitors. The aim of this study was the development of an efficient direct extraction method for foodborne viral RNA from bivalve molluscs. Using Mengovirus as a surrogate for foodborne viruses, five extraction methods based on RNA release by Trizol were compared on clams and oysters. A procedure consisting of Trizol, PureLink RNA Mini Kit, followed by Cetyltrimethylammonium bromide (CTAB) treatment and LiCl precipitation was found to provide RNA with the highest extraction efficiency and negligible inhibitory effect on real-time RT-PCR. This procedure was further compared to standard extraction method (ISO 15216) using clam, mussel and oyster samples spiked with Hepatitis A virus, Norovirus (NoV) GI and GII as well as bivalve samples naturally contaminated with NoV GI or GII. Results clearly demonstrated that the developed method provided, on average, a recovery 4·3 times higher than the standard reference protocol as well as good repeatability.
A direct extraction procedure was developed to recover viral RNA from shellfish with improved efficiency in comparison to reference extraction method (ISO 15216). Without the need for specific equipment, this procedure offers an alternative for performing food safety controls and for risk assessment studies. Given the inclusion in this extraction method of several steps for the efficient removal of food components inhibiting PCR reaction, this approach could serve as a general scheme for the extraction of nucleic acids of other enteric viruses and/or from other food categories.
由于双壳贝类中食源病毒浓度低且存在大量逆转录聚合酶链反应（RT-PCR）抑制剂，因此检测其中的食源病毒是一项具有挑战性的程序。本研究的目的是开发一种从双壳贝类中高效直接提取食源病毒RNA的方法。使用脑心肌炎病毒作为食源病毒的替代物，在蛤和牡蛎上比较了基于Trizol释放RNA的五种提取方法。发现一种由Trizol、PureLink RNA Mini试剂盒组成，随后进行十六烷基三甲基溴化铵（CTAB）处理和氯化锂沉淀的程序，能提供提取效率最高且对实时RT-PCR抑制作用可忽略不计的RNA。使用接种甲型肝炎病毒、诺如病毒（NoV）GI和GII的蛤、贻贝和牡蛎样本以及自然感染NoV GI或GII的双壳贝类样本，将该程序与标准提取方法（ISO 15216）进一步比较。结果清楚地表明，所开发的方法平均回收率比标准参考方案高4.3倍，且重复性良好。
开发了一种直接提取程序，与参考提取方法（ISO 15216）相比，能提高从贝类中回收病毒RNA的效率。该程序无需特定设备，为进行食品安全控制和风险评估研究提供了一种替代方法。鉴于此提取方法包含几个有效去除抑制PCR反应的食物成分的步骤，该方法可作为提取其他肠道病毒核酸和/或从其他食品类别中提取核酸的通用方案。
