A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food

Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination. The method exhibited good repeatability and stability in different thermal treatments and at ultra-high pressure. The relative standard deviation (RSD) values of 5% chicken content was less than 5.4% for ultra-high pressure or heat treatment. Moreover, we confirmed that different parts of meat had no effect on quantification accuracy of the ddPCR method. In contrast to real-time PCR, we examined the performance of ddPCR as a more precise, sensitive and stable analytical strategy to overcome potential problems of discrepancies in amplification efficiency discrepancy and to obtain the copy numbers directly without standard curves. The method and strategy developed in this study can be applied to quantify the presence and to confirm the absence of adulterants not only to sheep but also to other kinds of meat and meat products.肉类掺假是全球备受关注的食品安全问题。本文开发了一种全新的液滴数字PCR（droplet digital PCR，ddPCR）方法，用于定量检测绵羊与山羊肉制品中的鸡肉成分。与此同时，研究引入了一个常数（扩增倍数），将拷贝数比值转换为实际肉类成分占比。该ddPCR方法被证实比当前广泛应用于该检测领域的实时荧光定量PCR（real-time PCR）更为精准，在5%至80%的鸡肉含量范围内，其检测偏差小于9%。该方法在不同热处理及超高压条件下均表现出良好的重复性与稳定性；当鸡肉含量为5%时，超高压或热处理条件下的相对标准偏差（relative standard deviation，RSD）值均小于5.4%。此外，研究证实不同部位的肉类对该ddPCR方法的定量准确性无显著影响。与实时荧光定量PCR相比，本研究验证了ddPCR作为更精准、灵敏且稳定的分析策略的性能：它可克服扩增效率差异引发的潜在问题，且无需标准曲线即可直接获取拷贝数。本研究开发的方法与分析策略，不仅可用于绵羊源掺假成分的定量检测，还可推广至其他肉类及肉制品的掺假成分定量与阴性确认。