利用双重实时荧光重组酶辅助扩增技术快速检测肉制品中鼠源性成分

Rapid Detection of Mouse-Derived Components in Meat Products by Duplex Real-Time Recombinase-Aided Amplification

为实现肉制品中大鼠和小鼠源性成分的同步检测,基于实时荧光重组酶辅助扩增(real-time recombinase-aided amplification,Rt-RAA)技术,以大鼠COX3基因和小鼠16S rRNA基因序列保守区域为靶标,分别设计相应的Rt-RAA引物和探针,通过反应参数的优化确定最佳双重Rt-RAA检测反应条件。进一步对建立的双重Rt-RAA检测方法进行特异性、灵敏度及稳定性评估,并应用该方法对模拟市售肉制品进行检测。结果表明,所建立的双重RtRAA检测方法特异性强、稳定性好,大鼠和小鼠源性成分的检出限分别为0.5%和0.2%,同步检测2种源性成分检出限为0.5%,同步检测混合模拟市售样品检出限仍低至0.5%,双重Rt-RAA与实时荧光定量聚合酶链式反应对市售样品和模拟样品检测结果高度一致。因此,本研究建立的双重Rt-RAA检测方法适用于大鼠和小鼠源性成分的检测,为肉制品掺假检测提供了一种快捷、高效及准确的新方法。

In this study,real-time recombinase-aided amplification(Rt-RAA)was used for the synchronous detection of ratsand mice-derived components in meat products.Primers and probes targeting the conserved sequences of the COX3 gene in rats and those of the 16S rRNA gene in mice were designed.The reaction parameters were optimized to establish the optimal reaction conditions for duplex Rt-RAA detection.Furthermore,the specificity,sensitivity and stability of the established duplex Rt-RAA method were evaluated,and it was applied to simulated commercial meat products.The results showed that this method had strong specificity and stability.The detection limits for rats-and mice-derived components were 0.5%and 0.2%,respectively.The detection limits for their synchronous detection were both 0.5%,and the detection limits for synchronous detection of rats-and mice-derived components mixed in simulated commercial meat samples were still as low as 0.5%.Using duplex Rt-RAA and real-time fluorescence quantitative polymerase chain reaction(real-time PCR),highly consistent results were obtained for commercially available samples and simulated samples.Therefore,the duplex Rt-RAA method is suitable for the detection of rats-and mice-derived components in meat products,providing a fast,efficient,and accurate new method for the detection of meat product adulteration.