从生食鱼肉中提取GII型诺如病毒的不同方法的比较
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(上海市食品药品检验所,上海 201203)

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范一灵 男 主管药师 研究方向为食品和药品的微生物检验和方法 E-mail:tcfyl@163.com

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Comparison of different methods for extracting GII norovirus from raw fish
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(Shanghai Institute for Food and Drug Control,Shanghai 201203,China)

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    摘要:

    本研究比较了多种可能适用于生食水产鱼肉中诺如病毒(NoV)的提取方法。方法 人工污染GII型NoV三文鱼肉样品,通过病毒洗脱、浓缩、核酸提取,以及实时荧光定量反转录-聚合酶链式反应(RT-qPCR)检测等步骤的比较和优化,提高样品中NoV的检测成功率和回收率。结果 采用Tri/Glycine/Beef extract缓冲溶液洗脱、PEG/NaCl溶液沉降、增加氯仿-正丁醇(1∶1,V/V)溶液萃取等步骤,可以有效富集样品中的NoV,并有效去除样品中的PCR抑制成分。优化后的检测方案可以将人工污染GII型NoV的鱼肉样品的回收率由不足3%提高至15.15%,检测灵敏度可以达到17.3 RT-PCRU/25 g。结论 优化后的病毒提取方法具有良好的耐用性和重现性,适合于生食水产鱼肉中NoV的快速检测。

    Abstract:

    This study was aimed to compare several potentially reliable extraction methods for norovirus (NoV) in ready-to-eat raw fish meat. Methods Several methods of viral elution, viral concentration, RNA extraction and real-time reverse-transcription quantitative PCR (RT-qPCR) were compared and optimized by artificial contaminated NoV type GII in raw salmon sashimi, in order to improve the NoV detection. Results It was shown that viral particles could be well extracted and PCR inhibitors could be eliminated by a combination elution of Tri/Glycine/Beef extract solution, PEG/NaCl precipitation and chloroform-butanol (1∶1,V/V) extraction. In addition, the optimized extraction and detection method in this study could increase the average recovery rate of NoV type GII from no more than 3% to 15.15% in ready-to-eat raw fish meat, and the detection sensitivity was 17.3 RT-PCRU/25 g.Conclusion This optimal method has a better performance in repeatability and reproducibility, and it is suitable for rapid detection of NoV in ready-to-eat raw fish meat.

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范一灵,杨美成,杨燕.从生食鱼肉中提取GII型诺如病毒的不同方法的比较[J].中国食品卫生杂志,2016,28(6):753-758.

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  • 收稿日期:2016-08-11
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  • 在线发布日期: 2017-01-06
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