食品科学 ›› 2017, Vol. 38 ›› Issue (12): 240-245.doi: 10.7506/spkx1002-6630-201712037

• 安全检测 • 上一篇    下一篇

实时荧光定量RT-PCR法快速定量检测果蔬中星状病毒

马丹,魏海燕,徐蕾蕊,魏咏新,汪琦,张西萌,付溥博,刘莉,赵晓娟,曾静   

  1. 北京出入境检验检疫局检验检疫技术中心,北京 100026
  • 出版日期:2017-06-25 发布日期:2017-06-26
  • 基金资助:
    公益性行业(质检)科研专项(201410080)

Development of a Real-Time Fluorescent RT-PCR Assay for Quantitative Detection of Astrovirus in Fruits and Vegetables

MA Dan, WEI Haiyan, XU Leirui, WEI Yongxin, WANG Qi, ZHANG Ximeng, FU Pubo, LIU Li, ZHAO Xiaojuan, ZENG Jing   

  1. Inspection and Quarantine Technical Center, Beijing Enter-Exit Inspection and Quarantine Bureau, Beijing 100026, China
  • Online:2017-06-25 Published:2017-06-26

摘要: 目的:针对不同果蔬表面建立星状病毒富集与RNA提取方法,结合已报道的实时荧光定量逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)引物和探针,实现果蔬中星状病毒的高灵敏快速定量检测。方法:利用10 倍梯度稀释的星状病毒cRNA分子检测Ct值及其对应的初始浓度,构建标准曲线,为星状病毒的定量检测提供参考。将ISO/TS 15216-2:2013针对果蔬中诺如病毒和甲肝病毒RNA的提取方法用于星状病毒的检测,利用人工感染的大白菜和草莓样品,分析病毒回收率、灵敏度及检测重复性。最终通过60?份果蔬样品的检测验证该方法的实用性。结果:所建立的实时荧光定量RT-PCR方法扩增效率达95.9%,检测低限为5.6?拷贝/反应。人工感染的大白菜样品中病毒回收率在0.94%~9.63%之间,而人工感染草莓样品中病毒回收率最高达1.03%。对同一感染浓度的样品在不同时间的检测结果变异系数均小于2%。最终检出1?份来自北京农贸市场的草莓样品为星状病毒阳性,检测阳性率达1.67%。结论:建立的果蔬中星状病毒荧光RT-PCR定量检测方法快速、高效、灵敏,在食源性病毒的日常筛查和风险评估工作中具有重要的应用价值。

关键词: 星状病毒, 实时荧光定量RT-PCR, 果蔬, 定量检测

Abstract: Objective: Different procures for viral concentration and RNA extraction from fruits and vegetables were proposed taking into account the difference in surface structure between both materials and they were used to develop a sensitive and rapid real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) for the quantitative detection of astrovirus in fruits and vegetables using the primers and probes designed according to published sequences. Methods: A standard curve for quantitative detection of astrovirus was constructed by plotting the cycle threshold (Ct value) versus the starting concentration of 10-fold serially diluted cRNA. Then the method for norovirus and hepatitis A virus RNA extraction from fruits and vegetables described in the standard ISO/TS 15216-2:2013 was applied to detect astrovirus. The viral recovery rate, sensitivity and reproducibility of the assay were evaluated by using artificially contaminated cabbage and strawberry samples. Finally, 60 fruit and vegetable samples were tested to demonstrate the feasibility of this method. Results: The amplification efficiency of the real-time fluorescent RT-PCR was 95.9%, with a limit of detection (LOD) of 5.6 copies per reaction. The viral recovery rate of artificially contaminated cabbage samples was 0.94%?9.63%, compared to only 1.03% for the strawberries. Analysis of the artificially contaminated samples containing the same viral levels demonstrated high reproducibility with a coefficient of variation (CV) of less than 2%. Additionally, one strawberry sample collected from a retail market in Beijing was shown to be astrovirus-positive, and the detection rate was 1.67%. Conclusion: The developed method is rapid, efficient and sensitive for quantitative detection of astrovirus in fruits and vegetables, and will be a useful tool for routine screening and risk assessment of foodborne viruses.

Key words: astrovirus, real-time fluorescent RT-PCR, fruits and vegetables, quantitative detection

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