基于三重实时荧光聚合酶链反应构建黄牛和牦牛源性成分同步检测方法

Development of a method for simultaneous detection of cattle and yak-origin ingredients based on triplex real-time fluorescence polymerase chain reaction

目的建立一种拥有内源质控的三重实时荧光聚合酶链反应(polymerase chain reaction,PCR)技术,用以快速鉴定肉和乳中黄牛和牦牛的源性成分。方法首先对黄牛肉、牦牛肉、黄牛奶、牦牛奶等7种动物产品进行特异性检测,同时通过稀释梯度方法验证此方法的绝对检出限(limit of detection,LOD),最后通过黄牛和牦牛肉掺假模拟试验确定该方法的相对灵敏度。结果该方法的特异性强,能特异性地检测到来源于黄牛和牦牛肉和乳的DNA,稳定扩增的内源质控有效地避免了假阴性结果,本方法针对黄牛奶的绝对LOD为2.5×10^(-3)5.0×10‒3 ng,针对牦牛奶的绝对LOD为2.5×10^(-3)5.0×10^(‒2)ng,本方法对黄牛肉和牦牛肉混合肉的相对灵敏度可达0.1%牦牛肉。结论所建立的三重实时荧光PCR方法特异性强、灵敏度高,可实现黄牛和牦牛源性以及内源质控的同步检测,又能通过内源质控排除实验假阴性结果。

Objective To establish a triplex real-time fluorescence polymerase chain reaction(PCR)technique with endogenous control,and realize the rapid detection of the cattle and yak origin ingredients in meat and milk.Methods Firstly,the specificity of 7 kinds of animal products,including cattle meat,yak meat,cattle milk and yak milk,etc.was tested.Meanwhile,the absolute limit of detection(LOD)of the method was verified by radient dilution.Finally,the relative sensitivity of the method was determined by simulated adulteration test of cattle meat and yak meat.Results The method was highly specific and could only detect the DNA extracted from meat and milk of cattle and yak.The stable amplification of endogenous control effectively avoided false negative results.The absolute LOD of this method were 2.5×10^(-3)‒5.0×10^(-3)ng for cattle’s milk and 2.5×10^(-3)‒5.0×10^(‒2)ng for yak’s milk.The relative sensitivity of this method for mixed meat of yellow beef and yak could reach 0.1%yak.Conclusion The established triplex real-time fluorescence PCR method showed high specificity,high sensitivity,which can realize the simultaneous detection of cattle and yak provenance and endogenous control,and can exclude false negative results through endogenous quality control.