双重荧光PCR法快速检测饲料中牛羊源成分

The method of duplex fluorescence PCR for rapid detection of cattle-and sheep-derived ingredients in feedstuff

为了快速、灵敏检测反刍动物饲料中的牛羊源成分,试验采用双重荧光PCR方法,根据线粒体环氧酶2(Cox2)和细胞色素b(Cytb)基因,分别设计牛羊特异性引物序列,基于SYBR荧光PCR技术,分析熔解曲线,根据吸收峰的位置不同,定性识别牛羊源成分。结果表明:试验测得单重牛PCR产物熔解曲线吸收峰对应的熔解温度为(76.0±0.5)℃,羊PCR产物熔解曲线吸收峰对应的熔解温度为(81.5±0.5)℃,且双重体系吸收峰与单重对应的熔解温度吻合,说明试验可行。试验检测牛成分的灵敏度为0.01%,检测羊成分的灵敏度为0.1%。该方法可以同时定性检测饲料中牛羊源性成分,成本较低,节省时间,可以应用于高通量的实际检测样品,灵敏度较高。

To rapidly and sensitively detect cattle - and sheep - derived ingredients in ruminant feedstuff, a method of duplex real - time PCR was used to design the sequences of specific primers for cattle and sheep according to mitochondrial cyclooxygenase 2 （Cox2） and cytochrome b （Cytb）, respectively. The melting curves were analyzed based on SYBR fluorescence PCR technology, and then cattle- and sheep- derived ingredients were qualitatively identified according to different positions of the absorption peak. The results showed that the melting temperatures corresponding to the absorption peaks of melting curves of cattle and sheep singlet PCR products were （76.0 ± 0.5 ） and （81.5± 0.5 ）℃, respectively, while the absorption peak of duplex system was consistent with the melting temperatures corresponding to single system, indicating that the test was feasible. The detection sensitivity of the method for cattle - and sheep - derived ingredients was 0.01% and 0.1%, respectively. The results indicate that the method of duplex fluorescence PCR can simultaneously and qualitatively detect cattle - and sheep - derived ingredients in feedstuff, which has lower costs and can save time. The method can be applied to detect actual samples with high throughput, which has higher sensitivity.