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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参考文献(31)

[1]	SAFDAR M, JUNEJO Y, ARMAN K, et al. A highly sensitive and specific tetraplex PCR assay for soybean, poultry, horse and pork species identification in sausages: Development and validation[J]. Meat Science,2014,98(2):296−300. doi: 10.1016/j.meatsci.2014.06.006
[2]	ZHANG W J, XUE J H, et al. Economically motivated food fraud and adulteration in China: An analysis based on 1553 media reports[J]. Food Control,2016,67:192−198. doi: 10.1016/j.foodcont.2016.03.004
[3]	ALI A A, ALTEMIMI A B, ALHELFI N, et al. Application of biosensors for detection of pathogenic food bacteria: A review[J]. Biosensors,2020,10(6):58. doi: 10.3390/bios10060058
[4]	CASCELLA M, BIMONTE S, BARBIERI A, et al. Dissecting the mechanisms and molecules underlying the potential carcinogenicity of red and processed meat in colorectal cancer (CRC): An overview on the current state of knowledge[J]. Infectious Agents & Cancer,2018,13(1):3.
[5]	KAMRUZZAMANM, MAKINOY, OSHITAS. Non-invasive analytical technology for the detection of contamination, adulteration, and authenticity of meat, poultry, and fish: A review[J]. Analytica Chimica Acta,2015,853:19−29. doi: 10.1016/j.aca.2014.08.043
[6]	ZHENG X, LI Y, WEI W, et al. Detection of adulteration with duck meat in minced lamb meat by using visible near-infrared hyperspectral imaging[J]. Meat Science,2019,149:55−62. doi: 10.1016/j.meatsci.2018.11.005
[7]	KANE D E, HELLBERG R S. Identification of species in ground meat products sold on the US commercial market using DNA-based methods[J]. Food Control,2015,59:158−163.
[8]	杜鹏. “马肉风波”与欧盟肉制品安全监管制度[J]. 世界农业,2015(4):82−86, 168. [DU P. Horsemeat incident and EU meat product safety supervision system[J]. World Agriculture,2015(4):82−86, 168.
[9]	SUPIAN K. Cross-contamination in processing, packaging, storage, and transport in halal supply chain[J]. 2018: 309−321.
[10]	LIANOU A, PAPAKONSTANTINOU M, NYCHAS G, et al. Fraud in meat and poultry products[J]. Food Fraud,2021:85−108.
[11]	王建昌, 王金凤, 陈瑞春, 田振祥. 鸭肉冒充牛羊肉的分子生物学检测[J]. 肉类研究,2012,26(6):20−23. [WANG J C, WANG J F, CHEN R C, et al. Molecular biological detection of adulteration of mutton and beef with duck[J]. Meat Research,2012,26(6):20−23.
[12]	彭媛媛, 武煊, 陶晓奇. 实时荧光PCR技术定量检测肉类掺假的研究进展[J]. 食品与发酵工业,2019,45(15):279−287. [PENG Y Y, WU X, TAO X Q. Quantitative detection of meat adulteration by real-time fluorescent PCR[J]. Food and Fermentation Industries,2019,45(15):279−287. doi: 10.13995/j.cnki.11-1802/ts.020130
[13]	WILSON J M, PLATTS-MILLS T A E. Meat allergy and allergens[J]. Molecular Immunology,2018,100:107−112. doi: 10.1016/j.molimm.2018.03.018
[14]	SMITH R, MCELWEE G. The “horse-meat” scandal: Illegal activity in the food supply chain[J]. Supply Chain Management,2020,26(5):565−578.
[15]	孙亚波, 刘正伟, 吕丹娜, 等. 不同蛋白水平日粮对辽西驴肉质营养成分的影响[J]. 饲料研究,2021,44(18):96−100. [SUN Y B, LIU Z W, LÜ D N, et al. Effect of different protein levels rations on meat nutrition composition of Liaoxi donkeys[J]. Feed Research,2021,44(18):96−100. doi: 10.13557/j.cnki.issn1002-2813.2021.18.022
[16]	尤娟, 罗永康, 张岩春, 等. 驴肉主要营养成分及与其它畜禽肉的分析比较[J]. 肉类研究,2008(7):20−22. [YOU J, LUO Y K, ZHANG Y C, et al. Nutrition composition of donkey meat and comparison with other livestock and poultry meat[J]. Meat Research,2008(7):20−22. doi: 10.3969/j.issn.1001-8123.2008.07.008
[17]	海小, 刘国强, 罗建兴, 等. 基于TaqMan实时荧光PCR检测鲜肉及加工肉制品中的驴源性成分[J]. 肉类研究,2018,32(4):62−67. [HAI X, LIU G Q, LUO J X, et al. Identification of donkey-derived ingredients in raw and processed meats using taqman probe based real-time polymerase chain reaction[J]. Meat Research,2018,32(4):62−67.
[18]	KREUZ G, ZAGON J, BROLL H, et al. Immunological detection of osteocalcin in meat and bone meal: A novel heat stable marker for the investigation of illegal feed adulteration[J]. Food Additives & Contaminants,2012,29(5):716−726.
[19]	NURJULIANA M, CHE M Y, MAT H D. Rapid identification of pork for halal authentication using the electronicnose and gas chmmatography mass spectrometer with headspace analyzer[J]. Meat Science,2018,8(4):638−644.
[20]	CHIC C, SHOWP L, KUOM L, et al. Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes[J]. Journal of Chromatography B,2007,846(1/2):230−239.
[21]	KUMAR Y, KARNE S C. Spectral analysis: A rapid tool for species detection in meat products[J]. Trends in Food Science & Technology,2017,62:59−67.
[22]	BAIIIN N Z, VOGENSEN F K, KARLSSON A H. Species determination: Can we detect and quantify meat adulteration?[J]. Meat Science,2009,83(2):165−174. doi: 10.1016/j.meatsci.2009.06.003
[23]	MIGUEL A R, TERESA G, ISABEL G, et al. TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures[J]. Meat Science,2005,70(1):113−120. doi: 10.1016/j.meatsci.2004.12.005
[24]	KESMEN Z, GULLUCE A, SAHIN F, et al. Identification of meat species by TaqMan-based real-time PCR assay[J]. Meat Science,2009,82(4):444−449. doi: 10.1016/j.meatsci.2009.02.019
[25]	FANG X, ZHANG C. Detection of adulterated murine components in meat products by TaqMan real-time PCR[J]. Food Chemistry,2016,192:485−490. doi: 10.1016/j.foodchem.2015.07.020
[26]	CESARE C, MARCO D D, FEDERICA M. Development and validation of fast real-time PCR assays for species identification in raw and cooked meat mixtures[J]. Food Control,2012,23(2):400−404. doi: 10.1016/j.foodcont.2011.08.007
[27]	RAHARJO T J, NURYANTI I, PATRIA F P, et al. Mitochondrial ND-1 gene-specific primer polymerase chain reaction to determine mice contamination in meatball[J]. International Food Research Journal,2018,25(2):638−642.
[28]	中华人民共和国国家质量监督检验检疫总局. 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[S]. 北京: 中国标准出版社, 2017. General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. SN/T 3730.4-2013. Identification of domestic animal ingredient in food and feed-Part 4: Detection ofdonkey ingredient-real-time PCR method[S]. Beijing: China Standards Press, 2017.
[29]	陈笑云, 徐晓丽, 徐俊锋, 等. 鸭源性IL-2基因DNA标准物质及其用途, CN111088367A[P]. 2020 CHEN X Y, XU X L, XU J F, et al. Duck DERIVED IL-2 gene DNA standard material and its use, CN111088367A[P]. 2020.
[30]	CHEN C, CHEN J, ZHANG Y, et al. Quantitative detection of beef and beef meat products adulteration by the addition of duck meat using micro drop digital polymerase chain reaction[J]. Journal of Food Quality,2020,2020(6):1−8.
[31]	中华人民共和国农业农村部. NY/T 3309-2018 肉类源性成分鉴定实时荧光定性PCR法[S]. 北京: 中国标准出版社, 2017 Ministry of Agriculture and Rural Affairs of the People's Republic of China. NY/T 3309-2018. Identification of animal-derived materials in meat-Qualitative realtime PCR method[S]. Beijing: China Standards Press, 2017.