肉中猪源性成分Real-time PCR定量检测技术

doi:10.3864/j.issn.0578-1752.2023.01.012

摘要/Abstract

摘要：

【目的】建立一种快速、准确的肉中猪源性成分定量检测方法。【方法】首先从GenBank数据库中筛选猪特异性的微卫星DNA，根据微卫星DNA核酸序列设计引物，对常见10种动物基因组DNA进行PCR扩增，通过有无扩增产物判断筛选的微卫星DNA对猪源性成分的特异性。然后根据微卫星DNA核酸序列，设计特异性引物和探针，建立猪源性成分Real-time PCR检测方法，采用双标准曲线分别对猪源性成分和总动物源性成分进行定量，计算猪源性成分的百分含量。【结果】筛选到猪特异性微卫星DNA（Accession EF172428），根据其序列设计的引物SEQ-sus2-F/R只能从猪基因组DNA中扩增出目的条带，其他动物的基因组均无目的条带扩增。建立的Real-time PCR检测方法灵敏度为0.02 ng/25 μL反应体系。该方法能够准确检测出混合DNA样品中猪源性成分和混合肉样品中猪源性成分，百分误差分别约为1.32%和1.06%—7.12%。【结论】本研究利用Real-time PCR技术建立的定量猪源性成分的检测方法可以用来检测猪源性成分在混合样品中的百分含量。

关键词: 肉, 猪, 动物源性成分, Real-time PCR, 定量

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

翟晓虎, 李翎旭, 陈小竹, 蒋怀德, 贺卫华, 姚大伟. 肉中猪源性成分Real-time PCR定量检测技术[J]. 中国农业科学, 2023, 56(1): 156-164.

ZHAI XiaoHu, LI LingXu, CHEN XiaoZhu, JIANG HuaiDe, HE WeiHua, YAO DaWei. Quantitative Detection Technology of Porcine-Derived Materials in Meat by Real-time PCR[J]. Scientia Agricultura Sinica, 2023, 56(1): 156-164.

图/表 12

表1

图1

图2

图3

表2

表3

图4

表4

表5

图5

图6

表6

参考文献 24

[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

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	225	AF375760
SEQ-sus2-F	CTTCTTCCTCAGTGGTCGTG	262	EF172428
SEQ-sus2-R	GCAGCCTTACTTCGTTTCTC	262	EF172428
SEQ-sus2-probe-F	CACACAATGGGAATAAATTG	185	EF172428
SEQ-sus2-probe-R	GTCAGTCATGGTTCTCTA		EF172428
Sus-Taqman-probe	Cy5-CCTTCAAGCAGTGCAGCCTTAC-BHQ2		EF172428
SEQ-common296-F	CTGCTAAACAATCCAATAAAC	155	AB584373
SEQ-common296-R	GAGGTCTCCATTACTAATAGA		AB584373
Common-Taqman-probe	Texas red-TAACCTCTTGTCTCTTCGGCTGATG-BHQ2		AB584373

退火温度 Annealing temperature (℃)	扩增效率 Efficiency (%)	决定系数 Coefficient of correlation (R²)	最小Ct值 Ct_min	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

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 Ct_min	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

退火温度 Annealing temperature (℃)	扩增效率 Efficiency (%)	决定系数 Coefficient of correlation (R²)	最小Ct值 Ct_min	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

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 Ct_min	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