抑制剂去除对冷冻草莓中诺如病毒RT-PCR检测的影响

摘要

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.%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.