实时荧光PCR技术快速检测莲蓉制品中芸豆成分

A Real-Time PCR Assay for Rapid Detection of Kidney Bean Component in Lotus Seed Paste Product

目的:建立基于实时荧光聚合酶链式反应(polymerase chain reaction,PCR)技术检测莲蓉制品中芸豆成分的方法。方法:以芸豆pvsbe2基因高度保守区域设计特异性引物和探针,通过对莲子及其他富含淀粉类植物DNA进行扩增,以验证方法的特异性;以1 ng/μL的芸豆DNA进行系列稀释,确定此法检测灵敏度;对含有1%芸豆与莲子的混合样品的DNA模板进行10倍梯度稀释,确定重量检测灵敏度;并应用此方法和PCR方法对市场样品进行了检测,对建立的荧光PCR方法进行验证。结果:该检测方法具有高度特异性,与莲子及其他高淀粉植物无交叉反应;DNA质量浓度的检测灵敏度达到1 pg/μL,重量检测灵敏度可达0.01%。含芸豆成分的莲蓉月饼扩增阳性,检测结果与食品标签相符。结论:本研究建立的实时荧光PCR方法具有特异性强、灵敏度高、快速简便的特点,更适合莲蓉制品中芸豆成分的快速检测。

Objective： This study aimed to establish a real-time PCR method for rapid detection of kidney bean components in Lotus seed paste product. Methods： Specific primers and probes were designed based on the highly conserved region of the pvsbe2 gene of Phaseolus coccineus L. Specificity was confirmed by DNA amplification of lotus seeds and other starchrich plants. In addition, 1 ng/μL DNA of kidney bean was gradually diluted to determine its sensitivity. The DNA template of a mixture sample which contained 1% kidney bean and lotus seeds was 10-fold diluted to verify the weight sensitivity. And this method and PCR were applied to determine market samples for further validation. Results： This method had a high specificity which displayed no cross reaction with lotus seeds and other starch-rich plants. The sensitivity for detecting kidney bean DNA concentration and the proportion of kidney bean component were 1 pg/μL and 0.01%, respectively. The detection results indicated that positive amplification appeared in kidney bean present in lotus-seed-paste moon cake, which conformed to the food labels. Conclusions： The real-time fluorescent PCR method established in this study has the characteristics of high specificity and sensitivity and is suitable for fast detection of kidney bean component in lotus seed paste products.