基于微滴数字PCR阿胶定量检测方法的建立

Establishment of Quantitative Detection Method of Donkey-hide Gelatin Based on Droplet Digital PCR

为对阿胶及相关产品进行精确定量和纯度鉴定,本研究建立了一种基于微滴数字PCR(dd PCR)的阿胶定量检测方法。结果显示,在一定范围内阿胶质量与DNA含量以及DNA含量与DNA绝对拷贝数间均呈线性关系。以DNA含量作为换算值,确定出阿胶质量M阿胶(mg)与DNA绝对拷贝数C(copies/μL)的线性关系为M阿胶=0.13C+3.05。最终利用dd PCR检测样品中目的基因的绝对拷贝数来确定样品中的阿胶含量。准确性验证实验显示实测值与真实值基本保持一致,实测偏差最大仅为4.0%,这表明所建立的方法准确可靠。利用该方法对市场上不同品牌的8个阿胶和2个阿胶速溶粉进行检测,结果显示8个阿胶中,3款阿胶较纯,2款阿胶的阿胶含量较低,最低的仅为23.3%。而2款阿胶速溶粉的阿胶含量分别为44.8%和39.8%。以上表明该方法能有效对市售的阿胶产品进行定量检测和纯度鉴定。本实验建立的基于dd PCR的阿胶定量检测方法准确、高效、稳定,有较大的应用价值和应用前景,可有效预防阿胶掺假现象的发生。

A quantitative detection method based on droplet digital PCR（ dd PCR） technique was established to determine the purity of donkey-hide gelatin and its subsidiary products.The results showed that in a given range,the correlationship between raw gelatin weight and DNA concentration and between DNA concentration and target DNA copy number were both close to linear.Using DNA concentration as intermediate,the linear formula between raw gelatin weight M（ mg） and target DNA copy number C（ copies/μL）,M = 0.13 C ＋ 3.05,was calculated out.Thus,gelatin quantification was carried out in the samples by the formula between raw gelatin weight and target DNA copy number,which was calculated by dd PCR. Accuracy test showed that measured value was basically the same as the real value and the maximum deviation was just 4%,which indicates that the method was accurate and reliable.Using this method,8 gelatins and 2 gelatin instant powder in the market were tested.Results showed that,in 8 gelatins,3 were relatively pure and 2 of them had low gelatin content and the lowest was just 23.3%. The content of gelatin in 2 gelatin instant powder was 44.8% and 39.8%,respectively.This showed that this method could be used effectively in quantitative detection and purity identification for donkey-hide gelatin and its subsidiary products. The quantitative detection method based on dd PCR in this paper was accurate,efficient and stable.It had great application value and application prospect,which can effectively prevent the occurrence of gelatin adulteration.