Microsoft Word 胡智恺-二校出550

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1 6 2 Vol. 6 No Journal of Food Safety and Quality Feb., 2015 PCR 胡智恺, 宋丽萍 *, 姜洁, 薛晨玉, 郭淼, 王丹, 赵琳娜 (, ) 摘要 : 目的方法, DNA DNA, PCR 结果 PCR Ct DNA DNA 5%, 结论,, PCR, 关键词 : ; ; PCR; ; Real-time PCR method for quantitative detection of chicken blending in beef HU Zhi-Kai, SONG Li-Ping *, JIANG Jie, XUE Chen-Yu, GUO Miao, WANG Dan, ZHAO Lin-Na (Beijing Municipal Center for Food Safety Monitoring and Risk Assessment, Beijing , China) ABSTRACT: Objective To quantitatively analyze the beef and chicken in blending samples. Methods The primers were designed according to the sequence of the single copy gene. A series of genome DNA were used as standard curve in real-time PCR. The mathematical conversion parameters about the mass of meat and DNA were determined. The weight/weight equivalents of beef and chicken in four blending samples were analyzed using real-time PCR. Results The weight/weight equivalents of beef and chicken was determined by calculated using Ct, standard curve and the mathematical conversion parameters about the mass of meat and DNA. The results showed that the absolute error of detection with a tolerance of less than 5%. Conclusion Quantitative analyzing of the composition of the animal species in single component sample could be realized using real-time PCR by designing the primer according to the sequence of genome. This research could provide technical support for food safety. KEY WORDS: meat adulteration; quantitative analysis; real-time PCR; chicken; beef 基金项目 : ( ) Fund: Supported by Key Program for Beijing Municipal Science & Technology Commission (Multi Indicator Synchronous Screening Identification and Quantitative Research on the Plant Growth Regulator and Species Composition of Food) * 通讯作者 :,,, slp_0705@sina.com *Corresponding author: SONG Li-Ping, Senior Engineer, Beijing Municipal Center for Food Safety Monitoring and Risk Assessment, Shixing Street No.64, Shijingshan District, Beijing , China. slp_0705@sina.com

chicken blending in beef HU Zhi-Kai, SONG Li-Ping *, JIANG Jie, XUE Chen-Yu, GUO Miao, WANG Dan, ZHAO Lin-Na (Beijing Municipal Center for Food Safety Monitoring and Risk Assessment, Beijing 100041,

2 2, : PCR 引言,, PCR, [1-4],, [5-9],,,,, [1, 10-13],,, [14,15] Rene [16], PCR [17] β-actin,,,, 2 材料与方法 2.1 材料与仪器, () (: ) 20%: 80%, 40%: 60%, 60%: 40% 80%: 20% PCR (premix Ex Taq TM) TaKaRa 3K18 Sigma ; MyiQ PCR ; (Eppendorf Biophotometer Plus) Eppendorf 2.2 实验方法 DNA 提取 CTAB, min, 5000 r/min, 4, 5 min, ; /,, r/min, 4, 15 min, ;,, r/min, 4, 10 min, ;, min, r/min, 4, 10 min, ; 75%, 7500 r/min, 4, 5 min,, 200 μl DNA 标准曲线的绘制 50 ng 100 ng 150 ng 200 ng 250 ng 300 ng 350 ng DNA DNA PCR lg DNA, Ct, DNA DNA 实时荧光 PCR 扩增 , 5 -FAM 3 -TAMRA 1 ( ) 表 1 实验所用的引物和探针列表 Table 1 The sequence of primers and probe / (pmol) bp Beef F 10 GTAGGTGCACAGTACGTTCTGAAG Beef R 10 GGCCAGACTGGGCACATG 96 Beef Probe 10 CGGCACACTCGGCTGTGTTCCTTGCA Chicken F 10 CCAACATGCCTTTAAACCCTCA Chicken R 10 GGAACTGTAGCCTTCTGACTCG 76 Chicken Probe 10 TGCCTTTCCTTCCCCGCCAGTCTC : 5-3

3 PCR 20 μl, DNA 2 μl( DNA ), 2 Premix Ex Taq 10 μl, 0.5 μl(10 pmol/μl), 1 μl(10 pmol/μl) PCR s; 95, 5 s; 60, 30 s M=d DNA( 1), d, DNA ( d=m/dna) DNA 0.1 g 0.05 g, DNA, g, d =M /DNA =1120, d =M /DNA =170; 0.05 g, d =M /DNA =1190, d =M /DNA =226, d d, d =1155, d =198 3 结果与分析 3.1 引物及探针的特异性检测 PCR DNA, PCR 2, 牛肉和鸡肉 DNA 扩增标准曲线的绘制 PCR, DNA PCR, 1, DNA PCR Ct lg DNA, : Y=-4.19X+35.39; Y=-3.566X Y Ct, X Lg DNA 3.3 相同质量的牛肉和鸡肉所提出的 DNA 浓度差异 PCR, 1 DNA DNA Fig. 1 The DNA amplification standard curve : A, B Table 2 表 2 引物与探针的 DNA 扩增特异性验证 The DNA amplification results for testing the primers and probe (Ct) N/A N/A N/A N/A N/A N/A N/A (Ct) N/A N/A N/A N/A N/A N/A N/A : DNA N/A 40

2 牛肉和鸡肉 DNA 扩增标准曲线的绘制 PCR, DNA 1.2.3 PCR, 1, DNA PCR Ct lg DNA, : Y=-4.19X+35.39; Y=-3.566X+34.17 Y Ct, X Lg DNA 3.3 相同质量的牛肉和鸡肉所提出的 DNA 浓度差异 PCR, 1 DNA DNA Fig.

4 2, : PCR 553 表 3 Table 3 相同质量牛肉和鸡肉所提 DNA 的浓度 The DNA concentration extracted in the same quality of beef and chicken (g) DNA (ng/μl) D (μl) 检测样品中牛肉和鸡肉的所含百分比含量 DNA PCR DNA PCR 4, DNA ; 1 ;, 5%, 4 讨论, PCR,,, 5%,,,,,,,,,, 表 4 Table 4 混合肉样中牛肉和鸡肉含量的定量检测 Measured values of beef and chicken samples (g) ( %) 20% 80% 40% 60% 60% 40% 80% 20% DNA (ng) (g) ( %) 参考文献 [1] Rodriguez MA, Garcia T, Gonzalez I, et al. Quantitation of mule duck in goose foie gras using TaqMan real-time Polymerase Chain Reaction [J]. J Agric Food Chem, (6): [2] Dooley JJ, Paine KE, Garrett SD, et al., Detection of meat species using TaqMan real-time PCR assays [J]. Meat Sci, (3): [3] Chiappini B, Brambilla G, Agrimi U, et al. Real-time polymerase chain reaction approach for quantitation of ruminant-specific DNA to indicate a correlation between DNA amount and meat and bone meal heat treatments [J]. J AOAC Int, (5): [4] Wang TH, Chang YL, Peng HH, et al. Rapid detection of fetal aneuploidy using proteomics approaches on amniotic fluid supernatant [J]. Prenat Diagn, (7):

2.1 DNA PCR DNA 2.2.3 PCR 4, DNA ; 1 ;, 5%, 4 讨论, PCR,,, 5%,,,,,,,,,, 表 4 Table 4 混合肉样中牛肉和鸡肉含量的定量检测 Measured values of beef and chicken samples 1 2 3 4 (g) 0.02 0.08 0.

5 554 6 [5] Pray-Grant MG, Daniel JA, Schieltz Z, et al. Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation [J]. Nature, (7024): [6] Guo J, Guo A. Crossmodal interactions between olfactory and visual learning in Drosophila [J]. Science, (5732): [7] Elias M, Hill RI, Willmott KR, et al. Limited performance of DNA barcoding in a diverse community of tropical butterflies [J]. Proc Biol Sci, (1627): [8] Vences M, Thomas M, Thomas M, et al. Deciphering amphibian diversity through DNA barcoding: chances and challenges [J]. Philos Trans R Soc Lond B Biol Sci, (1462): [9] Armstrong KF, Ball SL. DNA barcodes for biosecurity: invasive species identification [J]. Philos Trans R Soc Lond B Biol Sci, (1462): [10] Robin ED, Wong R. Mitochondrial DNA molecules and virtual number of mitochondria per cell in mammalian cells [J]. J Cell Physiol, (3): [11] Brown WM, George M, Wilson AC. Wilson, Rapid evolution of animal mitochondrial DNA [J]. Proc Natl Acad Sci U S A, (4): [12] Thiebot MH. Some evidence for amnesic-like effects of benzodiazepines in animals [J]. Neurosci Biobehav Rev, (1): [13] Bellagamba F, Moretti VM, Comincini S, et al. Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA [J]. J Agric Food Chem, (8): [14] Fajardo V, Gonzalez I, Martin I, et al. Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures [J]. Meat Sci, (2): [15] Rodriguez MA, Garcia T, Gonzalez L, et al. TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures [J]. Meat Sci, (1): [16] Koppel R, Daniels M, Claudia Brunen-Nieweler. Multiplex real-time PCR for the detection and quantification of DNA from duck, goose, chicken, turkey and pork [J]. Eur Food Res Technol, 2013, 236(2): [17],,,. PCR [J]., 2014, 39(10): Song LP, Xue CY, Lu Y, et al. Detection and quantifi cation pork in sheep products using real-time PCR [J]. Food Sci Technol, 2014, 39(10): 作者简介 ( 责任编辑 : 白洪健 ) 胡智恺, 本科, 主要研究方向为食品安全 @163.com 宋丽萍, 副高级工程师, 博士, 主要研究方向为生物化学与分子生物学 slp_0705@sina.com

Limited performance of DNA barcoding in a diverse community of tropical butterflies [J]. Proc Biol Sci, 2007. 274(1627): 2881 2889. [8] Vences M, Thomas M, Thomas M, et al.

Microsoft Word - 4839-4843 20150924002-李建煌-二校全出

Microsoft Word - 4839-4843 20150924002-李建煌-二校全出 6 12 Vol. 6 No. 12 215 12 Journal of Food Safety and Quality Dec., 215 ONPG 李建煌 1, 姚蕾 1, 李玉优 1, 李焕仪 1, 何亮辉 1, 姜春宝 2, 曹维强 (1., 51852; 2., 5166) 1* 摘要 : 目的 βd(onitrophenylβdgalactopyranoside,

Microsoft Word 胡智恺-二校 出550

Microsoft Word 胡智恺-二校出550