牛肉及其中式加工品中猪肉成分的定性、定量检测方法研究
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收稿日期：2018-05-10；接受日期：2018-07-25 基金项目：国家自然科学基金（31372288）、陕西省科技统筹创新工程计划（2016KTCL02-36）、中央高校基本科研业务费专项（GK201805002） 联系方式：朱扬，E-mail：393857602@。通信作者刘永峰，E-mail：yongfeng200@
关键词：牛肉；猪肉；加工工艺；PCR；定性定量；灵敏度
Qualitative and Quantitative Detection Methods of Pork in Beef and Its Chinese Processing Products
ZHU Yang, LIU YongFeng, WEI YanChao, SHEN Qian, WANG YiFan
(College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062)
Abstract: 【Objective】 The objective of this paper was to establish a qualitative and quantitative detection method for pork components in beef and Chinese processed products, so as to guarantee the purity of beef products. 【Method】 The pig genomic
摘要：【目的】建立牛肉及中式加工品中猪肉成分的定性、定量检测方法，保障牛肉产品的纯正性。【方法】 提取猪肉及不同加工猪肉制品中的猪基因组 DNA，通过 DNA 质量检测，PCR 扩增，灵敏度试验，分析加工方式对猪 DNA 质量、灵敏度和检测限的影响；制备生牛肉和经干、蒸、煮、炖、煎、炸、烤制处理的牛肉制品中掺入不同 比例（10%、5%、1%、0.1%）猪肉的二元混合肉，进行普通 PCR 和荧光定量 PCR 定性定量检测，探索 DNA 在掺假鉴 别中的应用。【结果】不同加工方式下猪 DNA 质量检测结果显示，不同加工方式显著影响 DNA 纯度（P＜0.05），生 猪肉及 7 种猪肉制品中 DNA 纯度（A260nm/A280nm）范围为 1.893—1.977，高于理论值 1.8；DNA 含量范围为 110—277 µg·g-1， 经加工处理的猪肉制品的 DNA 含量显著高于生猪肉处理组（P＜0.05）；琼脂糖电泳结果发现，放置 6 个月后生猪 肉和 7 种肉制品的 DNA 严重降解，但生猪肉依然能获得一些不清晰的长片段 DNA，而 7 种肉制品的猪 DNA 全部降 解为小片段 DNA，说明长时间放置和热处理明显影响了猪 DNA 的完整性；猪肉制品中 DNA 的降解虽然严重，经普 通 PCR 扩增线粒体基因，所有样品的 PCR 产物均呈现为清晰且单一的条带，可见从加工肉制品中提取的 DNA 可以 开展灵敏度试验和掺假检测试验；灵敏度试验结果显示普通 PCR 是高度敏感的，经 10 倍梯度稀释，8 个试验组样 品中提取的猪 DNA 最低检测限均为 0.005 ng；荧光定量 PCR 扩增猪 DNA 所得 Ct 值形成的标准曲线也具有良好的 线性关系，其标准曲线斜率处在-3.1—-3.7，决定系数 R2 值均大于 0.99，PCR 扩增效率处在 89%—100%，且定量 PCR 最低能够检测出 0.005 ng 的猪 DNA。掺假样品定性定量 PCR 检测结果显示，除炸制混合肉（为 1%）外，混合 生肉及其他 6 种混合肉制品的定性检测试验最低检测限均为 0.1%，说明普通 PCR 可检测微量的猪肉成分；混合肉 的定量试验中根据不同掺假比例所建立的 8 个试验组标准曲线的决定系数 R2＞0.99，斜率为-3.1—-3.6，各曲线 均具有良好的线性关系，可以实现牛肉中猪肉成分的定量检测；对比生肉与肉制品的定量结果，混合生肉与混合 肉制品的标准曲线的截距之间存在约 0.01—0.6 个循环数的差异。【结论】不同加工处理能够显著影响肉中 DNA 的 含量、纯度和完整性，但不影响肉制品中 DNA 的检测限和灵敏度，普通 PCR 和定量 PCR 均可以检测到极微含量的 掺假肉成分。可见，基于 PCR 技术的检测方法灵敏度高、速度快、特异性强，定量检测标准曲线有较高的线性相 关性和扩增效率，可为肉类行业质量控制和检验计划以及验证标签声明提供可靠的依据，可应用于一些商业样品， 以保证肉制品的纯正性。
22 期
朱扬等：牛肉及其中式加工品中猪肉成分的定性、定量检测方法研究
4353
DNA from pork and different processed pork products were extracted, and then effects of processing methods on pig DNA quality, sensitivity and detection limit were analyzed through DNA quality testing, PCR amplification and sensitivity test. The raw beef was prepared, and the dried, steamed, boiled fried, stewed and roasted beef products were mixed with the binary mixed meat of different proportions (10%, 5%, 1%, 0.1%) of pork, and then the qualitative and quantitative detections of common PCR and real-time PCR were carried out. The application of DNA in adulteration identification. 【Result】 The DNA quality test results of different processing methods showed that different processing methods significantly affected the purity of DNA (P<0.05). The DNA purity (A260nm/A280nm) ranged from 1.893 to 1.977 in raw pork and seven kinds of pork products, which were higher than the theoretical value of 1.8. The DNA content ranged from 110 to 277 µg·g-1, and the DNA content of the processed pork products was significantly higher than that of the raw pork treatment group (P<0.05); Agarose electrophoresis showed that the DNA of raw pork and seven kinds of meat products was seriously degraded after 6 months of storage, but raw pork still obtained some unclear long-segment DNA, and the pig DNA of seven meat products all degraded into small fragments of DNA, indicating that long-term placement and heat treatment significantly affect the integrity of pig DNA; Although the degradation of DNA in pork products was serious, the mitochondrial genes were amplified by ordinary PCR, and the PCR products of all samples were presented as clear and single bands. It could be seen that the DNA extracted from the processed meat product could be tested for sensitivity and adulteration; The sensitivity test results showed that the common PCR was highly sensitive. The 10-fold gradient dilution showed that the minimum detection limit of pig DNA extracted from the eight test group samples was 0.005 ng. The standard curve formed by fluorescence quantitative PCR amplification of pig DNA was also formed, which had a good linear relationship. The slope of the standard curve was between -3.1 and -3.7, the coefficient of determination R2 was greater than 0.99, the PCR amplification efficiency was between 89% and 100%, and the quantitative PCR could be detected 0.005 ng of pig DNA. Qualitative quantitative PCR test results of adulterated samples showed that the minimum detection limit of qualitative test for mixed raw meat and other six mixed meat products was 0.1% except for fried mixed meat (1%), indicating that ordinary PCR could detect trace amounts of pork composition. In the quantitative test of mixed meat, the coefficient of determination (R2) of the standard curve of eight test groups established according to different adulteration ratios was more than 0.99, and the slope was -3.1--3.6. Each curve had a good linear relationship and could realize beef with quantitative detection of medium pork components. Comparing the quantitative results of raw meat and meat products, there was a difference of about 0.1 to 0.6 cycles between the intercepts of the standard curve of mixed raw meat and mixed meat products. 【Conclusion】 Different processing could significantly affect the content, purity and integrity of DNA in meat, but it did not affect the detection limit and sensitivity of DNA in meat products. Both ordinary PCR and quantitative PCR could detect the micro-content of adulterated meat. It could be seen that the detection method based on PCR technology had high sensitivity, high speed and high specificity, and the quantitative detection standard curve had high linear correlation and amplification efficiency, which could provide reliable quality control and inspection plan for meat industry and verification label declaration. The results could be applied to some commercial samples to ensure the purity of meat products.
中国农业科学 2018,51(22):4352-4363 Scientia Agricultura Sinica
doi: 10.3864/j.issn.0578-1752.2018.22.013
牛肉及其中式加工扬，刘永峰，魏燕超，申倩，王一凡
（陕西师范大学食品工程与营养科学学院，西安 710062）
关键词：牛肉；猪肉；加工工艺；PCR；定性定量；灵敏度
Qualitative and Quantitative Detection Methods of Pork in Beef and Its Chinese Processing Products
ZHU Yang, LIU YongFeng, WEI YanChao, SHEN Qian, WANG YiFan
(College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an 710062)
Abstract: 【Objective】 The objective of this paper was to establish a qualitative and quantitative detection method for pork components in beef and Chinese processed products, so as to guarantee the purity of beef products. 【Method】 The pig genomic
摘要：【目的】建立牛肉及中式加工品中猪肉成分的定性、定量检测方法，保障牛肉产品的纯正性。【方法】 提取猪肉及不同加工猪肉制品中的猪基因组 DNA，通过 DNA 质量检测，PCR 扩增，灵敏度试验，分析加工方式对猪 DNA 质量、灵敏度和检测限的影响；制备生牛肉和经干、蒸、煮、炖、煎、炸、烤制处理的牛肉制品中掺入不同 比例（10%、5%、1%、0.1%）猪肉的二元混合肉，进行普通 PCR 和荧光定量 PCR 定性定量检测，探索 DNA 在掺假鉴 别中的应用。【结果】不同加工方式下猪 DNA 质量检测结果显示，不同加工方式显著影响 DNA 纯度（P＜0.05），生 猪肉及 7 种猪肉制品中 DNA 纯度（A260nm/A280nm）范围为 1.893—1.977，高于理论值 1.8；DNA 含量范围为 110—277 µg·g-1， 经加工处理的猪肉制品的 DNA 含量显著高于生猪肉处理组（P＜0.05）；琼脂糖电泳结果发现，放置 6 个月后生猪 肉和 7 种肉制品的 DNA 严重降解，但生猪肉依然能获得一些不清晰的长片段 DNA，而 7 种肉制品的猪 DNA 全部降 解为小片段 DNA，说明长时间放置和热处理明显影响了猪 DNA 的完整性；猪肉制品中 DNA 的降解虽然严重，经普 通 PCR 扩增线粒体基因，所有样品的 PCR 产物均呈现为清晰且单一的条带，可见从加工肉制品中提取的 DNA 可以 开展灵敏度试验和掺假检测试验；灵敏度试验结果显示普通 PCR 是高度敏感的，经 10 倍梯度稀释，8 个试验组样 品中提取的猪 DNA 最低检测限均为 0.005 ng；荧光定量 PCR 扩增猪 DNA 所得 Ct 值形成的标准曲线也具有良好的 线性关系，其标准曲线斜率处在-3.1—-3.7，决定系数 R2 值均大于 0.99，PCR 扩增效率处在 89%—100%，且定量 PCR 最低能够检测出 0.005 ng 的猪 DNA。掺假样品定性定量 PCR 检测结果显示，除炸制混合肉（为 1%）外，混合 生肉及其他 6 种混合肉制品的定性检测试验最低检测限均为 0.1%，说明普通 PCR 可检测微量的猪肉成分；混合肉 的定量试验中根据不同掺假比例所建立的 8 个试验组标准曲线的决定系数 R2＞0.99，斜率为-3.1—-3.6，各曲线 均具有良好的线性关系，可以实现牛肉中猪肉成分的定量检测；对比生肉与肉制品的定量结果，混合生肉与混合 肉制品的标准曲线的截距之间存在约 0.01—0.6 个循环数的差异。【结论】不同加工处理能够显著影响肉中 DNA 的 含量、纯度和完整性，但不影响肉制品中 DNA 的检测限和灵敏度，普通 PCR 和定量 PCR 均可以检测到极微含量的 掺假肉成分。可见，基于 PCR 技术的检测方法灵敏度高、速度快、特异性强，定量检测标准曲线有较高的线性相 关性和扩增效率，可为肉类行业质量控制和检验计划以及验证标签声明提供可靠的依据，可应用于一些商业样品， 以保证肉制品的纯正性。
22 期
朱扬等：牛肉及其中式加工品中猪肉成分的定性、定量检测方法研究
4353
DNA from pork and different processed pork products were extracted, and then effects of processing methods on pig DNA quality, sensitivity and detection limit were analyzed through DNA quality testing, PCR amplification and sensitivity test. The raw beef was prepared, and the dried, steamed, boiled fried, stewed and roasted beef products were mixed with the binary mixed meat of different proportions (10%, 5%, 1%, 0.1%) of pork, and then the qualitative and quantitative detections of common PCR and real-time PCR were carried out. The application of DNA in adulteration identification. 【Result】 The DNA quality test results of different processing methods showed that different processing methods significantly affected the purity of DNA (P<0.05). The DNA purity (A260nm/A280nm) ranged from 1.893 to 1.977 in raw pork and seven kinds of pork products, which were higher than the theoretical value of 1.8. The DNA content ranged from 110 to 277 µg·g-1, and the DNA content of the processed pork products was significantly higher than that of the raw pork treatment group (P<0.05); Agarose electrophoresis showed that the DNA of raw pork and seven kinds of meat products was seriously degraded after 6 months of storage, but raw pork still obtained some unclear long-segment DNA, and the pig DNA of seven meat products all degraded into small fragments of DNA, indicating that long-term placement and heat treatment significantly affect the integrity of pig DNA; Although the degradation of DNA in pork products was serious, the mitochondrial genes were amplified by ordinary PCR, and the PCR products of all samples were presented as clear and single bands. It could be seen that the DNA extracted from the processed meat product could be tested for sensitivity and adulteration; The sensitivity test results showed that the common PCR was highly sensitive. The 10-fold gradient dilution showed that the minimum detection limit of pig DNA extracted from the eight test group samples was 0.005 ng. The standard curve formed by fluorescence quantitative PCR amplification of pig DNA was also formed, which had a good linear relationship. The slope of the standard curve was between -3.1 and -3.7, the coefficient of determination R2 was greater than 0.99, the PCR amplification efficiency was between 89% and 100%, and the quantitative PCR could be detected 0.005 ng of pig DNA. Qualitative quantitative PCR test results of adulterated samples showed that the minimum detection limit of qualitative test for mixed raw meat and other six mixed meat products was 0.1% except for fried mixed meat (1%), indicating that ordinary PCR could detect trace amounts of pork composition. In the quantitative test of mixed meat, the coefficient of determination (R2) of the standard curve of eight test groups established according to different adulteration ratios was more than 0.99, and the slope was -3.1--3.6. Each curve had a good linear relationship and could realize beef with quantitative detection of medium pork components. Comparing the quantitative results of raw meat and meat products, there was a difference of about 0.1 to 0.6 cycles between the intercepts of the standard curve of mixed raw meat and mixed meat products. 【Conclusion】 Different processing could significantly affect the content, purity and integrity of DNA in meat, but it did not affect the detection limit and sensitivity of DNA in meat products. Both ordinary PCR and quantitative PCR could detect the micro-content of adulterated meat. It could be seen that the detection method based on PCR technology had high sensitivity, high speed and high specificity, and the quantitative detection standard curve had high linear correlation and amplification efficiency, which could provide reliable quality control and inspection plan for meat industry and verification label declaration. The results could be applied to some commercial samples to ensure the purity of meat products.
中国农业科学 2018,51(22):4352-4363 Scientia Agricultura Sinica
doi: 10.3864/j.issn.0578-1752.2018.22.013
牛肉及其中式加工扬，刘永峰，魏燕超，申倩，王一凡
（陕西师范大学食品工程与营养科学学院，西安 710062）