基于便携式荧光定量PCR仪定量检测驴肉中掺假鸭肉

朱天园; 焦新雅; 程书梅

doi:10.13386/j.issn1002-0306.2022080139

基于便携式荧光定量PCR仪定量检测驴肉中掺假鸭肉

doi: 10.13386/j.issn1002-0306.2022080139

河北农业大学食品科技学院，河北保定 071001

详细信息

作者简介:
朱天园（1995−），女，硕士研究生，研究方向：食品工程，E-mail：1272948763@qq.com

通讯作者:
程书梅（1970−），女，博士，副教授，研究方向：食品微生物技术，E-mail：516089669@qq.com

中图分类号: TS207.3
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出版历程
- 收稿日期: 2022-08-15
- 刊出日期: 2023-05-01

Detecting Donkey Meat Adulterated with Duck Meat by Portable Fluorescence Quantitative PCR

College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China

摘要

摘要: 为建立快速方便的驴肉制品分子鉴定方法，本文以驴肉和常见的掺假肉类（鸭肉）为研究对象，筛选特异性引物和TaqMan探针，利用便携式Mini8 Plus实时荧光定量PCR仪进行灵敏度和特异性实验，通过绘制扩增标准曲线及确定驴肉和鸭肉的质量与DNA比值常数，对不同掺入比例（加入定量的鸭肉制成含量分别为20%、40%、60%、80%）的模拟样品和实际驴肉样品进行检测。结果显示，该方法对驴、鸭肉均具有良好的特异性，可以与马、猪、山羊、梅花鹿、牛、鸡、狗肉明显区分；对驴源性DNA成分的检出限为0.01 ng/μL，鸭源性DNA成分的检出限为0.1 ng/μL，对驴肉与鸭肉混合物中鸭肉成分的灵敏度为0.1%（w/w）；所建立的标准曲线线性关系良好，驴肉DNA扩增标准曲线：y=−3.584x+27.003，R²=0.9982；鸭肉DNA扩增标准曲线：y=−3.538x+30.907，R²=0.9991；采用已建立的方法对35份驴肉样本进行市场试点调查，发现6份（17.1%）驴肉样本中含有鸭肉成分。以上研究结果说明，该实时荧光定量PCR方法可用于驴肉产品中其他掺假肉类（鸭）的快速、准确检测，为驴肉及其制品的市场监管和相关执法提供有力的技术保障。
- 实时荧光PCR /
- 驴肉 /
- 鸭肉 /
- 肉制品掺假 /
- TaqMan
Abstract: To develop a fast and convenient method for the molecular identification of donkey meat products, this study selected donkey meat and duck meat (a common meat used for adulteration) as the research objects, screened specific primers and TaqMan probes, and conducted sensitivity and specificity experiments with a portable Mini8 Plus real-time fluorescence quantitative PCR instrument. By drawing the amplification standard curve and determining the mass/DNA ratio constants of donkey meat and duck meat, it detected the real donkey meat samples and the simulated samples adulterated with different proportions of duck meat (20%, 40%, 60%, and 80%, respectively). The results showed that the method had good specificity for both donkey meat and duck meat, which could be clearly distinguished from meat from horses, pigs, goats, sika deer, cattle, chickens, and dogs. The detection limit of donkey-derived DNA components was 0.01 ng/μL, and that of duck-derived DNA components was 0.1 ng/μL. Its sensitivity to duck meat components in the mixture of donkey meat and duck meat was 0.1% (w/w). The established standard curve showed a sound linear relationship. The amplification standard curve of donkey DNA was: y=−3.584x+27.003, R²=0.9982. The amplification standard curve of duck DNA was: y=−3.538x+30.907, R²=0.9991. A market pilot survey was conducted on 35 donkey meat samples using the established method, and revealed that 6 donkey meat samples (17.1%) contained duck meat. These results indicated that the real-time PCR method could be used for the rapid and accurate detection of other meat (such as duck meat) adulterated in donkey meat products. The combination of a simple detection procedure with a portable real-time PCR system provides technical support for food adulteration monitoring and control at the retail or market level.
- real-time fluorescence PCR /
- donkey meat /
- duck meat /
- adulteration of meat products /
- TaqMan

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图 1 驴（A）与鸭（B）的DNA扩增标准曲线

Figure 1. The DNA amplification standard curve of donkey (A) and duck (B)

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表 1 实验所用的引物和探针列表

Table 1. The sequence of primers and probes in the experiment

物种	标靶基因	引物和探针	扩增长度（bp）	参考文献
驴	ATPase6	F：AATAGCTCTAGCCGTACGGCTAACT R：CAGGATAATGAATGTAATAAGGGCTG P：TGCCGGACATCTTCTAATCCACCTT	130	SN/T 3730.4-2013食品及饲料中常见畜类品种的鉴定方法第4部分：驴成分检测实时荧光PCR法^[28]
鸭	IL-2	F：GGAGCACCTCTATCAGAGAAAGACA R：GTGTGTAGAGCTCAAGATCAATCCC P：TGGGAACAAGCATGAATGTAAGTGGATGGT	212	陈笑云等^[29]

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表 2 标准样品制备的DNA效果

Table 2. DNA effect of standard sample preparation

样本名称	A_{260 nm}/A_{280 nm}	DNA浓度（ng/μL）
驴	1.91	80.05
鸭	1.90	81.00
马	1.88	76.10
猪	1.89	73.55
山羊	1.80	75.45
牛	1.82	73.41
鸡	1.91	83.80
狗	1.86	85.30
梅花鹿	1.89	78.50

下载: 导出CSV

表 3 引物与探针DNA扩增特异性验证

Table 3. The DNA amplification results for testing the primers and probes

	驴	鸭	马	猪	山羊	梅花鹿	牛	鸡	狗
驴引物及探针（Ct）	25.40±0.33	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
鸭引物及探针（Ct）	N/A	31.89±0.24	N/A	N/A	N/A	N/A	N/A	N/A	N/A
注：N/A：未检测到驴、鸭的DNA。

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表 4 驴、鸭源性成分实时荧光PCR法检测限分析结果

Table 4. Results of real-time fluorescence PCR for detection of donkey and duck components

DNA浓度（ng/μL）	10	1	0.1	0.01	0.001
驴（Ct值）	20.78±0.11	24.42±0.01	30.09±0.13	36.81±0.03	N/A
鸭（Ct值）	26.35±0.02	29.91±0.08	35.20±0.15	N/A	N/A
注：N/A：未检测到驴、鸭的DNA。

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表 5 模拟掺假样品的配制信息

Table 5. Simulated adulterated sample preparation information

样品	掺假比例（%）		Ct平均值^c	结果判断
样品	驴^a	鸭^b	Ct平均值^c	结果判断
1	99.99	0.01	N/A	未检测到鸭肉成份
2	99.9	0.1	35.13±0.10	检测到鸭肉成份
3	99	1	33.07±0.22	检测到鸭肉成份
4	90	10	31.10±0.24	检测到鸭肉成份
注：a：驴肉在整个肉样本中的比例（w/w）；b：鸭肉在整个肉样中的比例（w/w）；c：Ct值是重复分析的平均值（n=3）；N/A：未检测到鸭的DNA。

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表 6 驴和鸭肉质量与DNA比值常数确定

Table 6. The mathematical conversion parameters about the mass of meat and DNA in donkey and duck

样品类别	驴肉		鸭肉
质量（g）	0.02	0.04	0.02	0.04
DNA（ng/μL）	21.4	43.1	476.4	211.2
d（10⁶ μL）	9.31×10⁻⁴		1.15×10⁻⁴

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表 7 混合肉样中驴肉和鸭肉含量的定量检测

Table 7. Quantitative determination of donkey meat and duck meat in mixed meat samples

	1		2		3		4
	驴肉	鸭肉	驴肉	鸭肉	驴肉	鸭肉	驴肉	鸭肉
理论质量（g）	0.02	0.08	0.04	0.06	0.06	0.04	0.08	0.02
理论混合比例（质量百分比%）	20	80	40	60	60	40	80	20
扩增结果（Ct）	22.09	21.15	21.19	21.57	20.62	22.28	20.19	23.59
DNA （ng/μL）	23.44	575.44	41.69	436.52	60.26	275.42	79.43	117.49
检测混合百分比例（质量百分比%）	25.62	74.38	44.67	55.33	64.91	35.09	85.11	14.89
绝对误差（%）	5.62	5.62	4.67	4.67	4.91	4.91	5.11	5.11

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表 8 市售驴肉制品的鸭肉成分荧光PCR检测结果

Table 8. Fluorescent PCR detection results of duck meat components in donkey meat products

样品种类	数量（份）	掺有鸭肉的样品数（份）	不合格率（份）
生鲜驴肉	25	4	17.1%
真空包装驴肉	10	2	17.1%

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