Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (1): 156-164.doi: 10.3864/j.issn.0578-1752.2023.01.012

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Quantitative Detection Technology of Porcine-Derived Materials in Meat by Real-time PCR

ZHAI XiaoHu1(), LI LingXu2(), CHEN XiaoZhu2, JIANG HuaiDe2, HE WeiHua1, YAO DaWei2()   

  1. 1. Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, Jiangsu
    2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095
  • Received:2022-03-26 Accepted:2022-11-01 Online:2023-01-01 Published:2023-01-17
  • Contact: YAO DaWei

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Abstract:

【Objective】The aim of this study was to develop a rapid and accurate quantitative method for identifying porcine-derived materials. 【Method】Porcine microsatellites DNA were selected from GenBank nucleotide database. Primers specific for porcine were designed based on the sequences of microsatellite DNA. Genomic DNA from 10 kinds of common animals was amplified by PCR method. The specificity of selected microsatellite DNA to porcine-derived materials was judged by the amplification products. According to the microsatellite sequence, the specific primers and probes were designed to establish a Real-time PCR method for identifying porcine-derived materials. The double standard curve was used to quantify the porcine-derived materials and total animal-derived materials, respectively, and the percentage content of porcine-derived materials was calculated. 【Result】Porcine specific microsatellite DNA with the accession number EF172428 was selected. Only porcine DNA gave a fragment through PCR assay, while there was no amplification for other non-target animal species DNA. The limit of detection was 0.02 ng in a 25 μL reactive system using the Real-time PCR method. This method could accurately detect porcine-derived components in mixed DNA samples and mixed meat samples with 1.32% percent error and 1.06%-7.12% percent error, respectively. 【Conclusion】The quantitative detection method of porcine-derived materials by Real-time PCR in this research could be used to detect the percentage content of porcine-derived materials in mixed samples.

Key words: meat, swine, animal-derived materials, Real-time PCR, quantitation

Table 1

Information of primers and Taqman probes"

引物名称 Primers name 序列 Sequence (5′-3′) 片段大小 Sizes (bp) 基因编号Accession
SEQ-sus1-F AACCCTGCCTGCCCTTTGT 225 AF375760
SEQ-sus1-R TGGCTCAGCGTCCATCCCT
SEQ-sus2-F CTTCTTCCTCAGTGGTCGTG 262 EF172428
SEQ-sus2-R GCAGCCTTACTTCGTTTCTC
SEQ-sus2-probe-F CACACAATGGGAATAAATTG 185 EF172428
SEQ-sus2-probe-R GTCAGTCATGGTTCTCTA
Sus-Taqman-probe Cy5-CCTTCAAGCAGTGCAGCCTTAC-BHQ2 EF172428
SEQ-common296-F CTGCTAAACAATCCAATAAAC 155 AB584373
SEQ-common296-R GAGGTCTCCATTACTAATAGA
Common-Taqman-probe Texas red-TAACCTCTTGTCTCTTCGGCTGATG-BHQ2 AB584373

Fig. 1

Specificity of porcine primers SEQ-sus1-F and SEQ- sus1-R 1: DNA Marker; 2: Pork; 3: Beef; 4: Goat; 5: Chicken; 6: Duck; 7: Goose; 8: Rabbit; 9: Donkey; 10: Horse; 11: Deer; 12: Blank control. The same as below"

Fig. 2

Specificity of porcine primers SEQ-sus2-F and SEQ- sus2-R"

Fig. 3

Specific detection of porcine primer and Taqman Dotted arrow: DNA form swine; Hollow arrow: DNA form other animals; Solid arrow: NTC"

Table 2

Annealing temperature optimization of porcine primer"

退火温度
Annealing temperature (℃)		 扩增效率
Efficiency (%)		 决定系数
Coefficient of correlation (R2)		 最小Ct值
Ctmin		 非特异性扩增Ct值
Non-specific amplification Ct		 空白对照Ct值
Blank control Ct
57.0 95.51±3.51 0.9987±0.0015a 21.75±0.73c 33.65±1.07c 未检测到Undetected
58.5 94.96±5.39 0.9990±0.0007a 22.40±0.31b 35.70±1.90b 未检测到Undetected
60.0 97.73±1.10 0.9979±0.0015a 22.71±0.37b 35.52±0.71b 未检测到Undetected
61.5 97.76±1.09 0.9909±0.0017b 24.47±0.80a 39.17±0.15a 未检测到Undetected

Table 3

Optimization of porcine primer and Taqman concentration"

探针-引物浓度
Probe and primer content (nmol·L-1)		 扩增效率
Efficiency
(%)		 决定系数
Coefficient of correlation (R2)		 最小Ct值
Ctmin		 非特异性扩增Ct值
Non-specific amplification Ct		 空白对照Ct值
Blank control Ct
150-150 91.18±5.55ab 0.9973±0.0018 23.55±0.50a 35.89±0.64bc 未检测到Undetected
150-250 95.37±0.63ab 0.9998±0.0001 22.56±0.31bc 34.77±0.10c 未检测到Undetected
150-300 96.08±3.81ab 0.9996±0.0001 22.52±0.28bc 35.28±0.13bc 未检测到Undetected
200-150 92.92±5.07ab 0.9970±0.0013 23.54±0.42a 35.03±0.82bc 未检测到Undetected
200-250 95.04±1.46ab 0.9989±0.0002 22.19±0.07c 37.85±0.35a 未检测到Undetected
200-350 97.31±1.80a 0.9993±0.0006 22.33±0.14c 34.57±0.66c 未检测到Undetected
250-150 97.71±0.86a 0.9943±0.0052 23.32±0.31a 36.09±0.35b 未检测到Undetected
250-250 95.58±2.37a 0.9985±0.0004 22.45±0.12bc 34.62±0.70c 未检测到Undetected
250-350 88.20±7.57b 0.9928±0.0080 22.83±0.68b 35.20±0.63bc 未检测到Undetected

Fig. 4

Real-time PCR amplification of animal-derived materials"

Table 4

Annealing temperature optimization of common primer"

退火温度
Annealing temperature (℃)		 扩增效率
Efficiency (%)		 决定系数
Coefficient of correlation (R2)		 最小Ct值
Ctmin		 空白对照Ct值
Blank control Ct
55.5 91.40±1.93ab 0.9963±0.0012 22.55±0.64d 未检测到Undetected
57.0 93.66±1.37ab 0.9969±0.0001 22.59±0.62d 未检测到Undetected
58.5 95.92±3.06a 0.9961±0.0019 22.849±0.65c 未检测到Undetected
60.0 96.77±1.86a 0.9951±0.0009 23.68±0.59b 未检测到Undetected
61.5 95.45±2.78a 0.9691±0.0296 26.70±0.96a 未检测到Undetected

Table 5

Optimization of common primer and TaqMan concentration"

探针-引物浓度
Probe and Primer (nmol·L-1)		 扩增效率
Efficiency
(%)		 决定系数
Coefficient of correlation (R2)		 最小Ct值
Ctmin		 非特异性扩增Ct值
Non-specific amplification Ct		 空白对照Ct值
Blank control Ct
150-150 95.8183±3.4270 0.9990±0.0001 22.07±0.20a 36.537950 未检测到Undetected
150-250 94.6394±0.2007 0.9983±0.0001 21.64±0.07b 36.072550 未检测到Undetected
150-300 96.1199±4.7783 0.9980±0.0017 21.60±0.11b 36.309250 未检测到Undetected
200-150 97.3010±0.2045 0.9979±0.0004 22.12±0.30a 37.725000 未检测到Undetected
200-250 97.0340±1.1993 0.9983±0.0007 21.70±0.13b 35.923600 未检测到Undetected
200-350 96.7049±3.9347 0.9967±0.0020 21.63±0.15b 36.617600 未检测到Undetected
250-150 97.1256±2.4211 0.9976±0.0011 21.97±0.42a 36.158100 未检测到Undetected
250-250 93.8683±6.0015 0.9952±0.0054 21.65±0.13b 36.344000 未检测到Undetected
250-350 94.6619±3.6286 0.9956±0.0059 21.56±0.15b 36.441800 未检测到Undetected

Fig. 5

Standard curve of porcine-derived genomic DNA"

Fig. 6

Standard curve of animal-derived genomic DNA"

Table 6

Results of porcine-derived composition detection in mixed meat samples"

实际值
Actual value
(%)		 检测值
Measured value
(%)		 百分误差
Percentage error (%)
样品1 Sample 1 40.00 42.20±1.66 5.50
样品2 Sample 2 70.05 70.79±11.06 1.06
样品3 Sample 3 63.96 60.10±10.60 1.34
样品4 Sample 4 60.34 64.64±15.36 7.12
[1]
史艳宇, 王莹, 石虹, 李天雨, 华蕾. 微滴数字PCR方法检测畜肉食品中鸭源性成分. 食品安全质量检测学报, 2018, 9(3): 583-588.
SHI Y Y, WANG Y, SHI H, LI T Y, HUA L. Detection of duck-derived materials in meat products by droplet digital PCR. Journal of Food Safety and Quality, 2018, 9(3): 583-588. (in Chinese)
[2]
SUL S, KIM M J, LEE J M, KIM S Y, KIM H Y. Development of a rapid on-site method for the detection of chicken meat in processed ground meat products by using a direct ultrafast PCR system. Journal of Food Protection, 2020, 83(6): 984-990. doi: 10.4315/jfp-19-583.

doi: 10.4315/JFP-19-583 pmid: 32034408
[3]
KIM M J, KIM H Y. A fast multiplex Real-time PCR assay for simultaneous detection of pork, chicken, and beef in commercial processed meat products. LWT-Food Science and Technology, 2019, 114: 108390. doi: 10.1016/j.lwt.2019.108390.

doi: 10.1016/j.lwt.2019.108390
[4]
陈念, 赖小平. 线粒体基因组: 结构特点和基因含量进化. 生物学杂志, 2011, 28(1): 70-73, 17. doi: 10.3969/j.issn.1008-9632.2011.01.070.

doi: 10.3969/j.issn.1008-9632.2011.01.070
CHEN N, LAI X P. Mt-genome revolution: Structure and gene content. Journal of Microbiology, 2011, 28(1): 70-73, 17. doi:10.3969/j.issn.1008-9632.2011.01.070. (in Chinese)

doi: 10.3969/j.issn.1008-9632.2011.01.070
[5]
陈传君, 金鹭, 林华, 胡滨, 韩国全, 陈世界, 张婧, 安微, 杨苗. 食品中羊肉源性成分微滴数字PCR定量方法的建立. 食品与发酵工业, 2020, 46(6): 229-237.
CHEN C J, JIN L, LIN H, HU B, HAN G Q, CHEN S J, ZHANG J, AN W, YANG M. Quantification of mutton-derived ingredients in food by droplet digital PCR. Food and Fermentation Industries, 2020, 46(6): 229-237. (in Chinese)
[6]
BALLIN N Z, VOGENSEN F K, KARLSSON A H. Species determination-Can we detect and quantify meat adulteration? Meat Science, 2009, 83(2): 165-174. doi: 10.1016/j.meatsci.2009.06.003.

doi: 10.1016/j.meatsci.2009.06.003
[7]
WANG Z C, WANG Z Y, LI T T, QIAO L, LIU R, ZHAO Y, XU Z Z, CHEN G, YANG S M, CHEN A L. Real-time PCR based on single- copy housekeeping genes for quantitative detection of goat meat adulteration with pork. International Food Science and Technology, 2020, 55(2): 553-558. doi: 10.1111/ijfs.14350.

doi: 10.1111/ijfs.14350
[8]
刘立兵, 陈敏娜, 孙晓霞, 张亦琴, 付琦, 钱云开, 周巍, 郭春海, 王建昌. 微滴式数字聚合酶链式反应对香肠制品中鸡、猪、牛源性成分的定量分析. 肉类研究, 2020, 34(8): 51-56.
LIU L B, CHEN M N, SUN X X, ZHANG Y Q, FU Q, QIAN Y K, ZHOU W, GUO C H, WANG J C. Quantitative analysis of chicken-, porcine- and bovine-derived ingredients in sausage products by droplet digital polymerase chain reaction. Meat Research, 2020, 34(8): 51-56. (in Chinese)
[9]
TABERLET P, WAITS L P, LUIKART G. Noninvasive genetic sampling: Look before you leap. Trends in Ecology and Evolution, 1999, 14(8): 323-327. doi: 10.1016/S0169-5347(99)01637-7.

doi: 10.1016/S0169-5347(99)01637-7 pmid: 10407432
[10]
SELKOE K A, TOONEN R J. Microsatellites for ecologists: A practical guide to using and evaluating microsatellite markers. Ecology Letters, 2006, 9(5): 615-629. doi: 10.1111/j.1461-0248.2006.00889.x.

doi: 10.1111/j.1461-0248.2006.00889.x pmid: 16643306
[11]
萨姆布鲁克 J, 拉塞尔 D W, 黄培堂. 分子克隆实验指南. 三版. 北京: 科学出版社, 2002.
SAMBROOK J, RUSSELL D W, HUANG P T. Molecular Cloning:A Laboratory Manual. 3rd ed. Beijing: Science Press, 2002. (in Chinese)
[12]
WU Z Z, GONG H F, ZHANG M P, TONG X K, AI H S, XIAO S J, PEREZ-ENCISO M, YANG B, HUANG L S. A worldwide map of swine short tandem repeats and their associations with evolutionary and environmental adaptations. Genetics Selection Evolution, 2021, 53(1): 39. doi: 10.1186/s12711-021-00631-4.

doi: 10.1186/s12711-021-00631-4
[13]
KRALIK P, RICCHI M. A basic guide to real time PCR in microbial diagnostics: Definitions, parameters, and everything. Frontiers in Microbiology, 2017, 8: 108. doi: 10.3389/fmicb.2017.00108.

doi: 10.3389/fmicb.2017.00108 pmid: 28210243
[14]
DOOLEY J J, PAINE K E, GARRETT S D, BROWN H M. Detection of meat species using TaqMan real-time PCR assays. Meat Science, 2004, 68(3): 431-438. doi: 10.1016/j.meatsci.2004.04.010.

doi: 10.1016/j.meatsci.2004.04.010 pmid: 22062411
[15]
MAYER W, HOCHEGGER R. Discrimination of two alleles of the melanocortin receptor 1 gene to discern European wild boar (Sus scrofa scrofa) and domestic pig (Sus scrofa domestica) in meat products by real-time PCR. European Food Research and Technology, 2011, 232(4): 687-692. doi: 10.1007/s00217-010-1402-8.

doi: 10.1007/s00217-010-1402-8
[16]
巫坚, 黄晓韵, 王海华, 陈小聪. 实时荧光PCR法检测肉制品中猪源性成分. 食品安全质量检测学报, 2021, 12(9): 3715-3720.
WU J, HUANG X Y, WANG H H. CHEN X C. Detection of porcine- derived components in meat products by real-time fluorescence PCR. Journal of Food Safety and Quality, 2021, 12(9): 3715-3720. (in Chinese)
[17]
CHEN X Y, LU L X, XIONG X H, XIONG X, LIU Y J. Development of a real-time PCR assay for the identification and quantification of bovine ingredient in processed meat products. Scientific Reports, 2020, 10(1): 2052. doi: 10.1038/s41598-020-59010-6.

doi: 10.1038/s41598-020-59010-6
[18]
LIU G Q, LUO J X, XU W L, LI C D, GUO Y S, GUO L. Improved triplex real-time PCR with endogenous control for synchronous identification of DNA from chicken, duck, and goose meat. Food Science and Nutrition, 2021, 9(6): 3130-3141. doi: 10.1002/fsn3.2272.

doi: 10.1002/fsn3.2272
[19]
LI J, GAO H F, LI Y J, XIAO F, ZHAI S S, WU G, WU Y H. Event-specific PCR methods to quantify the genetically modified DBN9936 maize. Journal of Food Composition and Analysis, 2022, 105: 104236. doi: 10.1016/j.jfca.2021.104236.

doi: 10.1016/j.jfca.2021.104236
[20]
LABRADOR M, GIMÉNEZ-ROTA C, ROTA C. Real-time PCR method combined with a matrix Lysis procedure for the quantification of listeria monocytogenes in meat products. Foods, 2021, 10(4): 735. doi: 10.3390/foods10040735.

doi: 10.3390/foods10040735
[21]
黄文胜, 邓婷婷, 韩建勋, 吴亚君, 陈颖. 转基因定量检测的不确定度研究. 中国生物工程杂志, 2012, 32(1): 49-55.
HUANG W S, DENG T T, HAN J X, WU Y J, CHEN Y. Estimate the uncertainty on quantification of GMO by the fluorescence real-time PCR method. China Biotechnology, 2012, 32(1): 49-55. (in Chinese)
[22]
REN Y F, LI X, LIU, Y M, YANG L T, CAI Y C, QUAN S, PAN L W, CHEN S S. A novel quantitative real-time PCR method for identification and quantification of mammalian and poultry species in foods. Food Control, 2017, 76: 42-51. doi: 10.1016/j.foodcont.2017.01.003.

doi: 10.1016/j.foodcont.2017.01.003
[23]
BEJERANO G, PHEASANT M, MAKUNIN I, STEPHEN S, KENT W J, MATTICK J S, HAUSSLER D. Ultraconserved elements in the human genome. Science, 2004, 304(5675): 1321-1325. doi: 10.1126/science.1098119.

doi: 10.1126/science.1098119 pmid: 15131266
[24]
WANG W J, WANG X Y, WEI T, ZHANG Q D, ZHOU X, LIU B. A multiplex Real-time PCR approach for identification and quantification of sheep/goat, fox and murine fractions in meats using nuclear DNA sequences. Food Control, 2021, 126: 108035. doi: 10.1016/j.foodcont.2021.108035.

doi: 10.1016/j.foodcont.2021.108035
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