一种牛肉检测的荧光实时定量方法研究

Study on a Fluorescence Real-time Quantitative Method for Beef Detection

[目的]开发一种用于牛源性成分检测的荧光实时定量检测方法。[方法]通过序列比对后,以牛源性线粒体DNA中cytb基因为模板,设计特异性的引物和探针,并以不同来源的DNA为模板,分析引物和探针的特异性。对牛源性DNA进行梯度稀释后,分析不同浓度下扩增曲线,分析检测方法的灵敏度。以牛源性DNA稀释液为模板,进行10个平行扩增试验,分析检测方法的精密度。[结果]该研究所用的引物和探针对牛源性DNA具有明显的扩增曲线,而对非牛源性(鸡、鸭、猪、人)DNA模板不进行扩增。灵敏度分析结果显示,该检测方法对浓度仅为24.4 pg/μL牛源性DNA模板仍进行阳性扩增。精密度分析显示,10个平行试验扩增曲线相似,Ct值的标准差(SD)仅为0.41,精密度(CV)也仅为1.6%。[结论]该研究开发的牛肉成分检测的荧光实时定量方法特异性高,仅对牛源性DNA发生扩增。同时该方法灵敏度也很好,检出限为24.4 pg/μL,精密度也很高,重复性良好,适用于市场上肉制品中牛源性成分的检测。

[Objective]To establish a real-time fluorescence quantitative detection method for the detection of bovine-derived components.[Method]After sequence alignment,specific primers and probe were designed based on the cytb gene of bovine mitochondrial DNA,and the specificity of primers and probe was analyzed with different DNA samples.The detection method was also analyzed with gradient dilution samples of bovine DNA.Ten parallel amplification experiments were carried out using bovine DNA dilution as template to analyze the precision of the detection method.[Result]The primers and probes used in this study had obvious amplification curves for bovine-derived DNA,but do not amplify non-bovine-derived(chicken,duck,pig,human)DNA templates.Sensitivity analysis results showed that the detection method still positively amplified bovine-derived DNA templates with a concentration of only 24.4 pg/μL.Precision analysis showed that the amplification curves of the 10 parallel experiments were similar,the standard deviation(SD)of the Ct value was only 0.41,and the precision(CV)was only 1.6%.[Conclusion]The fluorescence real-time quantitative method for beef component detection developed by this research has high specificity and only amplifies bovine-derived DNA.At the same time,the sensitivity of the method is very good,the detection limit is 24.4 pg/μL,the precision is also high,and the repeatability is good.It is suitable for the detection of bovine-derived components in meat products on the market.