多重RT-PCR法检测燕窝及其制品掺伪成分

Determination of adulterated components in edible bird’s nests and their products by multiplex RT-PCR

目的建立一种食用燕窝中主成分及掺伪成分的双重和三重实时荧光PCR检测方法。方法本研究通过合成金丝燕成分、银耳成分、红藻成分特异性的引物和探针,建立燕窝中主成分及掺伪成分的双重和三重实时荧光PCR检测体系,并设计相应的特异性实验保证检测体系的准确性,最后使用所建立的方法对市售样品进行检测并收集数据,验证多重实时荧光PCR检测方法的适用性。结果本方法可在1 d内完成样品的检测,建立的体系对金丝燕成分、银耳成分的检测灵敏度为0.1 ng/μL,红藻成分的检测灵敏度为0.5 ng/μL;燕窝中掺入银耳的检测限可达到0.1%;燕窝中掺入石花菜的检测限可达到0.5%。结论本研究建立的多重实时荧光PCR检测方法具有准确、灵敏度高、特异性好的优点,可用于燕窝及其制品中掺假物成分的准确定性检测。

Objective To establish a method for the determination of principal components and adulterated components in edible bird's nests by multiplex real-time PCR. Methods According to the highly conserved gene sequences, the specific primers and taqman probes were designed. Meanwhile, the stable duplex and triplex RT-PCR system were established for the detection of swiftlet, white fungus and red algea ingredients, and introduced into the specific test to guarantee the accuracy. Finally, the established method was used to detect the market samples and collect the data, and the applicability of multiple real-time fluorescence PCR detection methods would be verified to be feasible. Results The method could complete the detection of the sample in one day. The limits of detection of this system for DNA quality of swiftlet and white fungus components were respectively 0.1 ng/μL, and the limit of detection for the red algea ingredient was 0.5 ng/μL. The volume adulterated limit of detection for white fungus in swiftlet was 0.1%(V/V) in mixtures;and the limit of detection for red algea milk in swiftlet was 0.5%(V/V) in mixtures. Conclusion This multiplex real-time PCR assay has the advantages of accuracy, high sensitivity and good specificity, which is suitable for determination of principal components and adulterated components in edible bird's nests and their products.