实时荧光定量RT-PCR法快速定量检测果蔬中星状病毒  

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

作　　者：马丹[1] 魏海燕[1] 徐蕾蕊[1] 魏咏新[1] 汪琦[1] 张西萌[1] 付溥博[1] 刘莉[1] 赵晓娟[1] 曾静[1] MA Dan;WEI Haiyan;XU Leirui;WEI Yongxin;WANG Qi;ZHANG Ximeng;FU Pubo;LIU Li;ZHAO Xiaojuan;ZENG Jing(Inspection and Quarantine Technical Center, Beijing Enter-Exit Inspection and Quarantine Bureau, Beijing 100026, China)

机构地区：[1]北京出入境检验检疫局检验检疫技术中心,北京100026

出　　处：《食品科学》2017年第12期240-245,共6页Food Science

基　　金：公益性行业(质检)科研专项(201410080)

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c RNA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。Objective:Different procures for viral concentration and RNA extraction from fruits and vegetables wereproposed taking into account the difference in surface structure between both materials and they were used to develop asensitive and rapid real-time fluorescent reverse transcription-polymerase chain reaction(RT-PCR)for the quantitativedetection 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 of10-fold serially diluted cRNA.Then the method for norovirus and hepatitis A virus RNAextraction from fruits and vegetables described in the standard ISO/TS15216-2:2013was applied to detect astrovirus.Theviral recovery rate,sensitivity and reproducibility of the assay were evaluated by using artificially contaminated cabbage andstrawberry samples.Finally,60fruit and vegetable samples were tested to demonstrate the feasibility of this method.Results:The amplification efficiency of the real-time fluorescent RT-PCR was95.9%,with a limit of detection(LOD)of5.6copiesper reaction.The viral recovery rate of artificially contaminated cabbage samples was0.94%?9.63%,compared to only1.03%for the strawberries.Analysis of the artificially contaminated samples containing the same viral levels demonstrated highreproducibility with a coefficient of variation(CV)of less than2%.Additionally,one strawberry sample collected from aretail market in Beijing was shown to be astrovirus-positive,and the detection rate was1.67%.Conclusion:The developedmethod is rapid,efficient and sensitive for quantitative detection of astrovirus in fruits and vegetables,and will be a usefultool for routine screening and risk assessment of foodborne viruses.

关 键 词：星状病毒 实时荧光定量RT-PCR 果蔬 定量检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学]