红薯源性成分微滴式数字PCR的检测与定量分析

Detection and Quantification of Sweet Potato Components by Droplet Digital PCR

摘要:

本文根据红薯单拷贝物种基因β-淀粉酶基因（Ipomoea batatas beta-amylase gene，β-AMY）特异性片段设计引物组和探针，建立了红薯成分特异性定量检测的微滴数字PCR检测方法及β-AMY基因拷贝数浓度(copy number/μL)与红薯质量（mg）的线性关系，并对方法的特异性、灵敏度和适应性均进行了测试。结果显示建立的红薯成分数字PCR检测方法特异于红薯成分检测，在20 μL反应体系中定量下限（limit of quantitation，LOQ）和检测下限（limit of detection，LOD）分别为2.53 copies/μL和0.67 copies/μL。根据建立的拷贝数浓度（copy number/μL）与红薯质量（mg）的线性关系，对红薯质量百分比为5%和25%的样本进行定量检测，检测结果经换算得到的红薯含量分别为4.15%和21.33%，回收率分别为83.06%和85.31%，RSD值在6.33%~6.95%之间，表明本方法可对质量百分比为5%及以上的红薯成分进行准确定量，在定量检测方面具有较大应用潜力，可为市场监管提供了技术支持。

Abstract:

In this work, primers and probes were designed based on the specific fragment of Ipomoea batatas beta-amylase gene (β-AMY), and a droplet digital PCR method for detection of sweet potato was established. The specificity, sensitivity and adaptability of this method were tested. The results showed that the established method was specific to the detection of sweet potato components. The limit of quantitation (LOQ) and limit of detection (LOD) were 2.53 copies/μL and 0.67 copies/μL, respectively. According to the established linear relationship between copy number concentration (copy number/μL) and the sweet potato quality (mg), the quantitative analysis of 5% and 25% of the sweet potato mass percentage was performed, and the sweet potato content converted according to the linear relationship was 4.15% and 21.33%, respectively. The recovery rates were 83.06% and 85.31%, respectively, and the RSD values were between 6.33% and 6.95%. It showed that this method can accurately quantify sweet potato components with a mass percentage of 5% or more and it indicated that ddPCR method had strong potential in quantifying sweet potato. This method can provide technical support for market supervision of sweet potato and its products.