生物学杂志

生物学杂志

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牡蛎诺如病毒多样性研究中病毒富集方法的评估

  

  1. 1. 上海海洋大学 食品学院， 上海 201306；2. 农业部水产品贮藏保鲜质量安全风险评估实验室， 上海 201306
  • 出版日期:2019-06-18 发布日期:2019-06-18
  • 通讯作者: 喻勇新，博士，讲师，研究方向为食源性病毒学，E-mail: yxyu@shou.edu.cn
  • 作者简介:郭萍, 硕士研究生, 研究方向为食源性病毒学, E-mail:739380900@qq.com
  • 基金资助:
    国家自然科学基金（31601570）;教育部高等学校博士点基金（20133104110006）

The evaluation of virus enrichment methods for norovirus diversity in oysters

  1. 1.College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306；2. Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation, Ministry of Agriculture, Shanghai 201306, China
  • Online:2019-06-18 Published:2019-06-18

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摘要： 牡蛎作为诺如病毒的载体，含有几乎所有的GI型和大部分GII型诺如病毒。为了有利于牡蛎中诺如病毒多样性的研究，对4种常用于宏基因组测序中病毒富集方法（PEG6000沉淀法、超速离心法、过滤法和直接提取法）进行比较评估，将含有GII.4型诺如病毒基因的慢病毒（Lentivirus）加入阴性牡蛎中作为内参，利用反转录荧光定量PCR计算4种方法处理前后Lentivirus的数量，以回收率作为方法评估的指标，并以16S PCR来验证处理后微生物污染情况。重复本实验得到的两次结果均显示这4种方法回收率无明显差异，直接提取法回收率相对较高，但同时也具有较为严重的微生物污染。综合考虑其仍显示较好的优越性，因此直接提取方法更适用于后期牡蛎诺如病毒多样性研究中RNA的提取。

关键词: 诺如病毒, 牡蛎, 直接提取法, 多样性研究

Abstract: Oysters, the main vector of NoVs, contain almost all GI and most GII genogroup. In order to explore the diversity of the NoVs in oysters, this study aimed to evaluate four virus enrichment methods which were usually used in metagenomics study (PEG6000 precipitation method, ultracentrifugation method, filtration method and direct extraction method). Here, reverse transcription quantitative PCR (RT-qPCR) was adopted to calculate the Lentivirus (with GII.4 NoVs gene) copies before and after treated with the four methods, and recovery rate of Lentivirus was the evaluation indicator. Besides that, 16S rRNA PCR was conducted to compare the microorganism contamination. Both results showed that these four methods had no obvious difference, and the direct extraction method had relatively higher recycle rate and more serious microorganism pollution. It demonstrated direct extraction method has relatively good superiority, which is more suitable to extract RNA for NoVs diversity in oysters.

Key words: norovirus, oyster, direct extraction, diversity research

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