微滴式数字PCR检测冷冻草莓中GⅠ、GⅡ型诺如病毒

Detection of GⅠ,GⅡNorovirus in Frozen Strawberries with Droplet Digital PCR

目的:建立一种检测冷冻草莓中诺如病毒(GⅠ和GⅡ)的逆转录微滴数字PCR(RT-ddPCR)方法。方法:根据ISO标准选定检测引物,优化反应体系,退火温度,进行了方法学实验,建立了一种快速检测冷冻草莓中GⅠ和GⅡ亚型诺如病毒的新方法。结果:确定了数字PCR检测GⅠ型诺如病毒退火温度为56.5℃,GⅡ型诺如病毒退火温度为58.1℃。RT-ddPCR检测GⅠ质粒标准品标准曲线的R^2=0.9947,RT-ddPCR检测GⅡ质粒标准品标准曲线的R^2=0.9950,说明该方法具有良好的线性关系。与RT-qPCR灵敏度对比,RT-ddPCR法的灵敏度比RT-qPCR法高一个数量级。在检测范围内,最低检测限低至个位拷贝数。RSD最小为3.8%,表明该实验重复性良好。浓度较低100 copies/μL左右时,RT-ddPCR的重复性不佳。结论:本研究建立的诺如病毒数字PCR法具有特异性强、灵敏度高、检测限低等优点,可用于冷冻草莓中诺如病毒的定量检测。

Objective:To establish a reverse transcription microdroplet digital PCR(RT-ddPCR)method for the detection of norovirus(GI and GII)in foods.Methods:Select primers according to ISO standards,optimize the reaction system,and anneal the temperature.Methodological experiments established a new method for rapid and simultaneous detection of GI and GII subtype norovirus in food.Results:The digital PCR was used to detect the annealing temperature of GI type norovirus at 56.5℃,and the annealing temperature of GII type norovirus was 58.1℃.The R^2=0.9947 standard curve of GI plasmid standard was detected by RT-ddPCR,and the R^2=0.9950 of the standard curve of GII plasmid standard by RT-ddPCR showed that the method had a good linear relationship.Compared with the RT-qPCR sensitivity,the sensitivity of the RT-ddPCR method was an order of magnitude higher than that of the RT-qPCR method.Within the detection range,the minimum detection limit was as low as the unit of copies.The minimum RSD was 3.8%,indicating that the experiment was reproducible.Conclusion:The norovirus digital PCR method established in this study had the advantages of high specificity,high sensitivity and low detection limit.It could be used for the quantitative detection of norovirus in frozen strawberries.