基于RT-dd PCR技术的食品中诺如病毒定量检测

Quantitative Detection of Norovirus in Foods Using Reverse Transcriptase Droplet Digital Polymerase Chain Reaction

目的:构建基于逆转录微滴式聚合酶链式反应(reverse transcriptase droplet digital polymerase chain reaction,RT-ddPCR)技术的食品中诺如病毒(norovirus,NoV)定量检测方法。方法:筛选引物探针,建立并优化GI、GII型NoV和MS2噬菌体一步法RT-ddPCR检测体系,确定特异性。利用NoV RNA标准物质和MS2噬菌体RNA确认RT-ddPCR检测体系的定量限、准确度和重复性。制备人工阳性样品,分析MS2噬菌体对定量结果的影响。结果:3个RT-ddPCR检测体系具有良好的特异性、准确度和重复性,NoV RT-ddPCR检测体系的定量限包含10~4~10~0拷贝/μL 5个数量级,MS2噬菌体RT-ddPCR检测体系定量限与NoV相当,添加与不添加MS2噬菌体的人工阳性样品的定量结果无显著差异(P≥0.05)。结论:NoV RT-ddPCR检测体系可准确定量NoV RNA,MS2噬菌体不影响定量结果,可作为过程控制的模式病毒被添加到食品中,指示回收率,计算食品中NoV实际载量。

Objective:To develop a quantitative detection method for norovirus(NoV)in foods based on digital PCR.Methods:Three sets of specific primers and probes were selected to establish one-step reverse transcriptase droplet digital polymerase chain reaction(RT-ddPCR)assays for NoV genotype I(GI),genotype II(GII)and MS2 phage,respectively.The specificity of these RT-ddPCR assays was verified.Their limit of quantitation,accuracy and repeatability were analyzed using NoV RNA reference materials and MS2 phage RNA.Artificially positive samples were prepared to analyze the influence of MS2 phage on the results of NoV detection.Results:The three RT-ddPCR assays had satisfactory specificity,accuracy and repeatability.The limit of quantitation of NoV RT-ddPCR was at five orders of magnitude from 104 to 100 copies/μL and close to that of MS2 phage RT-ddPCR.There was no significant difference between the quantitative results for artificially positive samples with and without added MS2 phage(P≥0.05).Conclusion:NoV RNA could be accurately quantified by the NoV RT-ddPCR assay.MS2 phage did not affect the quantitative results of NoV,and could be added as a process control model virus to indicate virus recovery and calculate the content of NoV particles in foods.