抑制剂去除对冷冻草莓中诺如病毒RT-PCR检测的影响

doi:10.7506/spkx1002-6630-201704048

食品科学 ›› 2017, Vol. 38 ›› Issue (4): 296-300.doi: 10.7506/spkx1002-6630-201704048

抑制剂去除对冷冻草莓中诺如病毒RT-PCR检测的影响

徐文斐，房保海，林超，王宫璞，王群，郑小龙，梁成珠，岳志芹

山东出入境检验检疫局检验检疫技术中心，山东青岛 266002

出版日期:2017-02-25 发布日期:2017-02-28
基金资助:
青岛市民生科技计划项目（14-2-3-60-nsh）；科技部质检公益项目（201410080-2）

Influence of PCR Inhibitor Removal on Detection of Norovirus GⅡ in Frozen Strawberry by Real-Time Fluorescent RT-PCR XU Wenfei, FANG Baohai, LIN Chao, WANG Gongpu, WANG Qun, ZHENG Xiaolong

XU Wenfei, FANG Baohai, LIN Chao, WANG Gongpu, WANG Qun, ZHENG Xiaolong, LIANG Chengzhu, YUE Zhiqin

Technique Center of Inspection and Quarantine, Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao 266002, China

Online:2017-02-25 Published:2017-02-28

摘要/Abstract

摘要： 目的：设计对比实验，以冷冻草莓为材料，研究样品中所含抑制剂对诺如病毒荧光定量逆转录聚合酶链式反应（reverse transcription polymerase chain reaction，RT-PCR）检测结果的影响，优化提高检测灵敏度与准确率。方法：GⅡ型诺如病毒便悬液的制备与RNA提取，已知阴性冷冻草莓样品进行前处理、病毒富集、病毒RNA提取纯化，分组（对照组、实验组），抑制剂去除，荧光定量RT-PCR检测。结果：随着病毒浓度的梯度递减，抑制剂影响作用呈明显增强趋势，导致在高于正常检测最低限度两个数量级的水平，即出现假阴性结果；抑制剂去除的实验组检测结果，随着病毒浓度梯度的递减明显优于未经处理的对照组；抑制剂去除的实验组稳定检出限为10－6（Ct值小于40），最低检出限为10－7（诺如病毒（norovirus，NoV）浓度梯度检测最低检出限为10－5）。结论：抑制剂去除可明显优化冷冻草莓中NoV荧光定量RT-PCR检测，提高检测方法的灵敏度，且操作简便快捷，检测成本浮动不大，期望应用于日常检测工作中。

关键词: 冷冻草莓, 诺如病毒, 荧光定量RT-PCR, 抑制剂

Abstract: Objective: Comparative experiments were designed to address the influence of the presence of PCR inhibitor on the results of detection of norovirus GⅡ in frozen strawberry by real-time fluorescent RT-PCR (RT-qPCR), aiming to improve the detection sensitivity and accuracy. Methods: After pretreatment of fecal suspensions containing norovirus GⅡ and known negative strawberry samples, the virus was enriched for the extraction and purification of RNA and a control group and an experimental group were established. After removal of the PCR inhibitor, the samples were determined by RTqPCR. Results: The effect of the inhibitor tended to increase obviously with decreasing concentration gradient of virus, thus giving false negative results which were two orders of magnitude higher than the lowest level detected without the presence of the inhibitor. Better detection results were obtained by removal of the inhibitor compared with the control untreated group, as the virus concentration gradient decreased. The stable detection limit (LOD) for the experimental group without PCR inhibitor was 10-6 (Ct value < 40), and the minimum LOD were 10-7. Conclusion: The detection of norovirus in frozen strawberries by fluorescence quantitative RT-PCR could be improved in terms of sensitivity by removal of the inhibitor. In addition, the improved method was quick, easy to operate, economic and practical.

Key words: frozen strawberry, norovirus G Ⅱ, RT-PCR, inhibitors

中图分类号:

X954

徐文斐，房保海，林超，王宫璞，王群，郑小龙，梁成珠，岳志芹. 抑制剂去除对冷冻草莓中诺如病毒RT-PCR检测的影响[J]. 食品科学, 2017, 38(4): 296-300.

XU Wenfei, FANG Baohai, LIN Chao, WANG Gongpu, WANG Qun, ZHENG Xiaolong, LIANG Chengzhu, YUE Zhiqin. Influence of PCR Inhibitor Removal on Detection of Norovirus GⅡ in Frozen Strawberry by Real-Time Fluorescent RT-PCR XU Wenfei, FANG Baohai, LIN Chao, WANG Gongpu, WANG Qun, ZHENG Xiaolong[J]. FOOD SCIENCE, 2017, 38(4): 296-300.

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抑制剂去除对冷冻草莓中诺如病毒RT-PCR检测的影响

Influence of PCR Inhibitor Removal on Detection of Norovirus GⅡ in Frozen Strawberry by Real-Time Fluorescent RT-PCR XU Wenfei, FANG Baohai, LIN Chao, WANG Gongpu, WANG Qun, ZHENG Xiaolong

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