6 2 Vol. 6 No. 2 2015 2 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 (, 100041) 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 100041, 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 :,,, E-mail: 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 100041, China. E-mail: slp_0705@sina.com
2, : PCR 551 1 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 2.2.1 DNA 提 取 CTAB, 65 30 min, 5000 r/min, 4, 5 min, ; /,, 12000 r/min, 4, 15 min, ;,, 12000 r/min, 4, 10 min, ;, -20 10 min, 12000 r/min, 4, 10 min, ; 75%, 7500 r/min, 4, 5 min,, 200 μl DNA 2.2.2 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 2.2.3 实 时 荧 光 PCR 扩 增 2.2.3.1, 5 -FAM 3 -TAMRA 1 ( 1 5-3 ) 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
552 6 2.2.3.2 PCR 20 μl, DNA 2 μl( DNA ), 2 Premix Ex Taq 10 μl, 0.5 μl(10 pmol/μl), 1 μl(10 pmol/μl) 2.2.3.3 PCR 95 30 s; 95, 5 s; 60, 30 s 40 2.2.3.4 M=d DNA( 1), d, DNA ( d=m/dna) DNA 0.1 g 0.05 g, 1.3.1 DNA, 3 0.1 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, 1.2.3 PCR 2, 1 3.2 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. 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 27.01 N/A N/A N/A N/A N/A N/A (Ct) N/A N/A N/A 27.63 N/A N/A N/A N/A : DNA N/A 40
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) 0.1 88.7 0.05 41.9 0.1 580.5 0.05 220.4 1155 198 3.4 检 测 样 品 中 牛 肉 和 鸡 肉 的 所 含 百 分 比 含 量 4 2.2.1 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 0.04 0.06 0.06 0.04 0.08 0.02 ( %) 20% 80% 40% 60% 60% 40% 80% 20% DNA (ng) 19.99 371.53 33.11 309.03 44.67 147.91 56.23 89.12 (g) 0.023 0.074 0.038 0.061 0.052 0.029 0.065 0.018 ( %) 23.7 76.3 38.3 61.7 64.2 35.8 78.3 22.7 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, 2004. 52(6): 1478 1483. [2] Dooley JJ, Paine KE, Garrett SD, et al., Detection of meat species using TaqMan real-time PCR assays [J]. Meat Sci, 2004. 68(3): 431 438. [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, 2005. 88(5): 1399 1403. [4] Wang TH, Chang YL, Peng HH, et al. Rapid detection of fetal aneuploidy using proteomics approaches on amniotic fluid supernatant [J]. Prenat Diagn, 2005. 25(7): 559 566.
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