为了提高检测效率,节省时间、试剂和样本,建立了基于高分辨率熔解曲线(high-resolution melting, HRM)的双重环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术用于羊乳中牛乳成分的检测。通过引物设计优化,牛引物目标产物的Tm值在80 ℃,羊引物目标产物的Tm值在83 ℃,由于在传统熔解曲线中峰型有交叉重叠,同时检测存在干扰,因此使用HRM技术,实现了2种不同动物源性成分的有效区分。该方法特异性好,不需要针对牛、羊源性成分单独进行检测,当体系中存在其中一种或两种都存在时都可将其快速检出,且对其他物种没有交叉反应及假阳性现象的发生,成功应用于市售羊乳及其制品的检测。
To improve the detection efficiency and decrease the cost, a duplex LAMP-HRM (loop mediated isothermal amplification-high resolution melting) method was established for the detection of cow milk components in goat milk. Through the optimization of primer design, the optimized Tm value of cow and goat amplicons were 80 ℃ and 83 ℃, respectively. The two melting peaks were overlap in traditional melting curve assay, but can be easily distinguished by the proposed LAMP-HRM method. This method could simultaneously detect the cow and goat components, and there was no cross-reactivity and false positives for other species. Moreover, the adulteration ratio could be quantified by the height of the melting peak, and it is applied to the detection of goat milk and its dairy products.
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