实时荧光PCR技术快速检测莲蓉制品中芸豆成分

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。

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

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 confirmedDNA amplification of lotus seeds and other starch-rich 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 suifor fast detection of kidney bean component in lotus seed paste products.