Quantitative species determination based on real time PCR–Can the results be expressed as weight/weight equivalents?

Food adulteration is a common challenge in the meat industry. Polymerase chain reaction (PCR) has been used as a method to detect contamination from different species of meat. From a consumer perspective, a PCR method with measurements in terms of weight/weight (w/w) ratios will be more familiar. In this study, the focus was on how to convert the results of quantitative analysis from genome/genome (g/g) to w/w using real-time PCR. The mixtures with different ratios of mutton in pork were analyzed as test samples. The c values of different species, as a reflection of the key conversion factors, were established and evaluated. The effects of heat treatment on w/w conversion of PCR data were also assessed. The results indicated that the c value shows significant variability among individual samples. An average c value was found to cause a bias of more than 7% for mixtures in the range of 20–80%. For individual meat samples with pre-determined c-values, real-time PCR was useful for quantitative analysis of mutton contamination in pork within the range of 20–80%, with a bias of detection of less than 2%. However, this method was shown to have a limit of quantification of 5% with mutton in pork. Furthermore, heat treatment (121°C, 15 min) significantly reduced the accuracy of quantitative analyses. Because the c value is not available for most commercial samples, and some food products are subjected to heat treatment as a method of sterilization, accurate quantitative analysis (w/w) may not be an option for commercial samples using PCR-based technology.

肉制品行业中，食品掺假是一项普遍存在的难题。聚合酶链式反应（Polymerase Chain Reaction，PCR）已被应用于检测不同物种来源肉类的掺假污染。从消费者视角出发，采用质量比（weight/weight，w/w）作为计量形式的PCR检测方法更为大众所熟知。本研究聚焦于如何利用实时荧光定量PCR（real-time PCR），将基因组拷贝数比（genome/genome，g/g）的定量分析结果转换为质量比（w/w）。研究以猪肉中掺入不同比例羊肉的混合样本作为测试样品，确立并评估了不同物种的c值——该值可反映核心转换因子的特性，同时评估了热处理对PCR数据转换为w/w结果的影响。研究结果显示，不同个体样本间的c值存在显著差异：当混合样本中羊肉占比处于20%~80%区间时，采用平均c值会导致超过7%的检测偏差。对于预先确定了c值的单种肉类样本，实时荧光定量PCR可有效实现20%~80%羊肉掺假比例下猪肉中羊肉含量的定量检测，其检测偏差小于2%。不过该方法针对猪肉中羊肉掺假的定量限为5%。此外，热处理（121℃、15分钟）会显著降低定量分析的准确性。由于多数商业食品样本无法获取c值，且部分食品需通过热处理实现灭菌，因此基于PCR技术的商业样本难以实现准确的质量比（w/w）定量分析。