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基于芯片式数字PCR的奶茶中牛乳成分定量分析
张梦姣1，2，3，王巍1，2，3，段庆梓1，2，3，孙宏虎1，2，3，王义1，2，3，毕小玲1，2，3，杨小琪1，2，3，尚柯1，2，3*
(1.成都市食品检验研究院，四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用），北京 100029；3.辐照保藏四川省重点实验室，四川 成都 611130)
Quantitative Analysis of Bovine Milk Ingredients in Milk Tea Relying on Chip-Based Digital PCR
ZHANG Mengjiao1，2，3，WANG Wei1，2，3，DUAN Qingzi1，2，3，SUN Honghu1，2，3，WANG Yi1，2，3，BI Xiaoling1，2，3，YANG Xiaoqi1，2，3，SHANG Ke1，2，3*
(1.Chengdu Institute of Food Inspection，Chengdu 611130，Sichuan，China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation，Beijing 100029，China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi)
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投稿时间：2024-10-14
中文摘要: 为检测现制奶茶中牛乳成分含量，依据一定范围内牛乳蛋白质质量浓度与DNA浓度、DNA浓度与拷贝数之间的线性关系，构建了一种基于芯片式数字聚合酶链式反应（chip-based digital polymerase chain reaction，cdPCR）技术的定量检测方法。对常见奶牛品种的Cyt b保守区域进行比对分析，设计特异性引物及探针，并筛选奶茶中牛乳成分DNA提取方法。经cdPCR检测后建立DNA浓度与拷贝数之间的线性关系。结果表明，设计的引物探针能特异性扩增市面上荷斯坦牛、娟姗牛、三河牛、西门塔尔牛4种常见奶牛品种；DNA提取试剂盒为奶茶中牛乳成分DNA基因组最佳提取方法；建立的cdPCR方法可检测牛奶中牛乳成分DNA浓度范围为4.25×100～4.49×104拷贝∕μL。该研究采用的cdPCR在其动态范围内R2=0.999 8，表现出良好的线性，且样本相对误差范围<20%。对50批市售奶茶样本进行检测，蛋白质质量浓度小于1 μg∕μL的样本数量占比60%，该方法为检测市售奶茶中的牛乳成分含量提供了一种准确的定量方法。
Abstract:To detect the content of bovine milk ingredients in freshly made milk tea，a quantitative detection method based on chip-based digital polymerase chain reaction （cdPCR） was constructed according to the linear relationships between bovine milk protein mass and DNA concentration and between DNA concentration and copy number within a certain range.Sequence alignment and analysis were performed on the conserved region of Cyt b from common dairy cattle breeds to design specific primers and probes and to screen the optimal DNA extraction method for bovine milk ingredients in milk tea.The linear relationship between DNA concentration and copy number was subsequently established via cdPCR detection.The results showed that the designed primers and probes could specifically amplify the target fragments from four common commercial dairy cattle breeds，namely Holstein cattle，Jersey cattle，Sanhe cattle，and Simmental cattle.The DNA extraction kit method was determined as the optimal method for genomic DNA extraction from bovine milk ingredients in milk tea.The established cdPCR method achieved a detectable range of 4.25×100-4.49×104 copies∕μL for bovine milk-derived DNA.The cdPCR method used in this study exhibited excellent linearity within its dynamic range （R2=0.999 8），and the quantification precision of all tested samples was below 20%.Detection of 50 batches of commercial milk tea samples revealed that 60% of the samples had a protein mass concentration lower than 1 μg∕μL.In conclusion，the method established in this study provides an accurate quantitative approach for the detection of bovine milk ingredient content in commercial milk tea.
文章编号：202609018 中图分类号： 文献标志码：
基金项目:四川省市场监督管理局科技计划项目（SCSJS2023002）
| 作者 | 单位 |
| 张梦姣 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 王巍 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 段庆梓 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 孙宏虎 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 王义 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 毕小玲 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 杨小琪 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| 尚柯 | 1.成都市食品检验研究院,四川 成都 611130；2.国家市场监管重点实验室（营养与健康化学计量及应用）,北京 100029；3.辐照保藏四川省重点实验室,四川 成都 611130 |
| Author Name | Affiliation |
| ZHANG Mengjiao | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| WANG Wei | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| DUAN Qingzi | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| SUN Honghu | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| WANG Yi | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| BI Xiaoling | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| YANG Xiaoqi | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
| SHANG Ke | 1.Chengdu Institute of Food Inspection,Chengdu 611130,Sichuan,China；2.Key Laboratory of Chemical Metrology and Applications on Nutrition and Health for State Market Regulation,Beijing 100029,China；3 I di ti P ti K L b t f Si h P i Ch d 611130 Si h Chi |
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