摘要
We present a red deer-specific real-time PCR assay which, combined with a reference real-time PCR assay published previously, allows the quantification of the red deer content in food products. Thus, it can be applied to detect food adulteration. The primer/probe system of the red deer-specific real-time PCR assay amplifies a 87 bp long fragment of the protein kinase C iota gene. To eliminate cross-reactivity with closely related species, the forward primer was designed to contain one deliberate base mismatch adjacent to one red deer-specific base. The red deer-specific real-time PCR assay did not show cross-reactivity with 23 animal and 50 plant species tested. LOD and LOQ, determined by analyzing a serially diluted DNA extract containing 1% (w/w) red deer DNA in pig DNA, were 0.05% and 0.4%, respectively. The accuracy was validated by analyzing DNA mixtures, meat extract mixtures, meat mixtures and model game sausages with known red deer content. The highest accuracy was obtained when the calibration mixture was similar to the analyzed sample in both the composition and concentration of the animal species of interest. High recoveries were not only obtained for raw samples but also after subjection to thermal treatment, including brewing (15 min at 75–78 °C), boiling (90 min at 100 °C) and microwave treatment (15 s, 40 s or 2 min at 650 W). The red deer-specific real-time PCR assay was found to be robust with respect to small deviations in the reaction volume or the annealing temperature and the use of another real-time PCR instrument.
摘要译文
我们提供了一种红鹿特异性实时PCR检测方法，结合之前发布的参考实时PCR检测方法，可以对食品中的红鹿含量进行定量。从而，它可以应用于检测食品掺假。红鹿特异性实时PCR测定的引物/探针系统扩增蛋白激酶C iota基因的87bp长片段。为了消除与密切相关物种的交叉反应性，正向引物设计为在一个红色鹿特异性碱基附近包含一个故意的碱基错配。红鹿特异性实时PCR测定法未显示与23种动物和50种测试植物物种的交叉反应性。 LOD和LOQ，通过分析在猪DNA中含有1％（w / w）马鹿DNA的连续稀释的DNA提取物而测定，分别为0.05％和0.4％。通过分析DNA混合物验证了准确性，肉提取物混合物，肉类混合物和具有已知马鹿含量的模型游戏香肠。当校准混合物与感兴趣的动物物种的组成和浓度两者中的分析样品相似时获得最高的准确度。高回收率不仅在原始样品中获得，而且在经受热处理之后也获得，包括酿造（15分钟75-78℃），沸腾（90分钟100℃）和微波处理（15秒，40秒或2分钟650W）。发现红鹿特异性实时PCR检测方法对于反应体积或退火温度的小偏差以及使用另一种实时PCR仪器而言是稳健的。
MariaKaltenbrunner[a][b];RupertHochegger[a];MargitCichna-Markl[b];. Red deer (Cervus elaphus)-specific real-time PCR assay for the detection of food adulteration[J]. Food Control, 2018,89: 157-166