Microsoft Word 史艳宇-确定版面.doc

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1 4 6 Vol. 4 No Journal of Food Safety and Quality Dec., 2013 PCR 史艳宇 1*, 刘金华 2, 吴月丹 3, 王莹 1, 王颖 1, 王潇 1, 刘晓晖 1, 周亮 1, 赵立群 (1., ; 2., ; 3., ) 1 摘要 : 目的方法, PCR, ; ; 50 mg/kg DNA DNA, 结果,, ( 0.1 μg/kg) 结论,, 关键词 : ; ; real time PCR; Rapid detection of duck-derived materials in meat products by real time PCR SHI Yan-Yu 1*, LIU Jin-Hua 2, WU Yue-Dan 3, WANG Ying 1, WANG Ying 1, WANG Xiao 1, LIU Xiao-Hui 1, ZHOU Liang 1, ZHAO Li-Qun 1 (1. Jilin Product Quality Supervision Inspection, Changchun , China; 2. Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun , China; 3. Jilin agricultural radio and Television School, Shulan , China) ABSTRACT: Objective To establish a rapid, specific, sensitive method for detection of duck derived materials. Methods The real-time PCR method for detection of duck-derived materials in meat products was established using forward and reverse primers and probe corresponding to the mitochondrial gene. The specific detection was carried out by livestock and poultry meat including mutton, beef, chicken, goose, porcine, rabbit meat, horse meat, venison and other reference animal species. Sensitivity detection was done using the 50 mg/kg mutton DNA as diluent for gradient dilution of duck DNA. Results This method can effectively detect duck derived materials, with a strong specificity and high sensitivity (up to 0.1 μg/kg), and without obvious influence by mutton components on duck sensitivity detection. Conclusion This method has a strong specificity and high sensitivity, and can rapidly and accurately detect the duck-derived components in meat products. KEY WORDS: duck-derived materials; mitochondrion gene; real time PCR; detection 基金项目 : (QK ) Fund: Supported by the General Administration of Quality Supervision, Inspection and quarantine of the People s of Republic of China (QK ) * 通讯作者 :,, E mail: shiyanyu219@163.com *Corresponding author: SHI Yan-Yu, Associate Professor, Jilin Product Quality Supervision Inspection, No.1088, Dongnanhu Road, Nanguan District, Changchun , China. E mail: shiyanyu219@163.com

2 引言,,,,,,,,, [1] [2-8],,,, 2 材料与方法 2.1 材料与试剂 ( [9] ) ; ; 2.2 主要仪器 (BECKMAN, ); (IKA M20, IKA Labortechnik, Staufen, ); PCR (ABI 7500 ); DNA/RNA/ (Bio Specmini, ); (0.1~ μl JILSON, ); 2.3 方法 [2] 样品前处理,, : 500 mg 50 ml, 2,, r/min 5 min, 2,, r/min 5 min, 1,, 2 ml, r/min 5 min,, r/min 5 min, DNA 的提取 DNA, DNA CTAB DNA: 200 mg 1.5 ml, 600 μl CTAB, 15 μl K, min; 500 μl - - (25:24:1, v/v), r/min 15 min;, r/min 10 min, ; 70% 2~3, ; 200 μl TE (TE DNA ); - (24:1, v/v) DNA DNA 引物设计 GenBank (accession KF156760) Primer Express 3.0, GenBank( BLAST), Blast,, Forword primer: 5 AAG CCT TCC TCT AGC TCA GC-3, Reverse primer: 5 -AGA AAA TGC TTT AGT TAA GTC-3, Probe: 5 -FAM-CTC AGC CGC TTA AAC AAC GC-TAMRA 反应体系和反应条件 : 2 TaqMan Universal PCR Master Mix 12.5 μl, 10 μmol/l 1 μl, DNA 1 μl, ddh 2 O 8.5 μl : 50 2 min; 95 5 min, s, s, 特异性实验灵敏度实验 DNA(50 mg/kg) DNA 10, DNA µg/kg DNA, PCR,

3 6, : PCR 荧光 PCR 方法用于实际食品样品检测, DNA, PCR 荧光 PCR 方法用于检测加工肉制品中鸭源性成分,, DNA, PCR 3 结果与分析 3.1 引物特异性验证 PCR, DNA PCR,,,,, 灵敏度实验 DNA DNA PCR, : 0.1 µg/kg, DNA 肉卷和肉板中的鸭源性成分检测 4 PCR, 市售加工肉制品中鸭源性成分检测,, DNA PCR,, PCR 4 1 PCR Fig. 1 Real time PCR result of specificity test

4 1862 食品安全质量检测学报图 2 荧光 PCR 敏感性检测结果 Fig. 2 Sensitivity detection of real time PCR Fig. 3 图 3 检测样品中的鸭源性成分 Determination of duck-derived materials from sold products 第4卷

5 6, : PCR 1863 Fig. 4 4 Determination of duck-derived materials from processed products 4 讨论,,,,,,,, DNA PCR( ) DNA,, PCR,, [10-16], PCR,,,,, 50 mg/kg DNA DNA, : DNA, 0.1 μg/kg,,, DNA, DNA,, [17-18],,,

1864 4 参考文献 [1]. [J]., 2012(8): 46-47. Zheng B. Hang sheep head sell duck [J]. Prosecutorial View, 2012(8): 46-47. [2] SN/T 2557-2010 PCR [S].

SN/T 2727-2010 Deteumination of poultry derived materials in feedstuff-real-time fluorescent PCR method[s]. [4] GB/T 21106-2007 PCR [S].

GB/T 21104-2007 Identification of Ruminantia derived materials (bovidia, caprinae, cervus) in animal-originated feedsatuffs -PCR method[s]. [6] GB/T 21105-2007 PCR [S].

6 参考文献 [1]. [J]., 2012(8): Zheng B. Hang sheep head sell duck [J]. Prosecutorial View, 2012(8): [2] SN/T PCR [S]. SN/T Detection of qualitation of bovine ingredient in meat product- Real time PCR method[s]. [3] SN/T PCR [S]. SN/T Deteumination of poultry derived materials in feedstuff-real-time fluorescent PCR method[s]. [4] GB/T PCR [S]. GB/T Identification of cervus derived materials in animal-originated feedsatuffs product-pcr method[s]. [5] GB/T ( ) PCR [S]. GB/T Identification of Ruminantia derived materials (bovidia, caprinae, cervus) in animal-originated feedsatuffs -PCR method[s]. [6] GB/T PCR [S]. GB/T Identification of Canis derived materials in animal-originated feedstuffs product-pcr method[s]. [7] SN/T PCR [S]. SN/T Determination of bovine, ovine, porcine derived materials in food, cosmetic and feed-real time PCR method [S]. [8] GB/T PCR [S]. GB/T Identification of Porcine derived materials in animal-originated feedstuffs-pcr method [S]. [9],,,. [M]. :, 1979, Zheng ZX, ZHANG YS, TANG ZZ, et al. Chinese animal Aves Volume II anseriformes [M]. Beijing: Science press, 1979, [10],,,. [J]., 2005, 25(5): 3 6. Zong H, Zeng SL, Lin QY, et al. Molecular biology techniques for identification of horse and donkey materials in feedstuff [J]. China Herbivores, 2005, 25(5): 3 6. [11] Yang BH, Zong H. Determination of animal-derived materials with conserved mitochondrial sequence in feedstuffs[j]. Feed Res, 2002, 5: 1 3. [12],,,. [J]., 2002, 38(1): 3 5. Yang BH, Zong H, Lin QY, et al. Identification of bovine and ovine derived materials in animal originated feedstuffs by molecular biological method[j]. Chin J Anim Sci, 2002, 38(1): 3 5. [13] Bottero MT, Dalmasso IA, Nucera D, et al. Development of a PCR assay for the detection of animal tissue in ruminant feeds[j]. J Food Prot, 2003, 66(12): [14] Martín I, García T, Fajardo Vk, et al. SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs[j]. Meat Sci, 2009, 82: [15],,,. PCR [J]., 2011,2(6): Liu SS, Li H, Luo SZ, et al. Progress in meat adulteration identification using PCR method [J]. Journal of Food Safety and Quality, 2011,2 (6): [16] SOARES S, AMARAL J S, MAFRA I, et al. Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay [J]. Meat Science, 2010, 85 (3) : [17] Fajardo V, González I, Martín 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, 2008, 79(2): [18] Fajardo V, González I, Martín I, et al. Real-time PCR for quantitative detection of chamois (Rupicapra rupicapra) and pyrenean ibex (Capra pyrenaica) in meat mixtures [J]. J AOAC Int, 2008, 91(1): 作者简介 ( 责任编辑 : 赵静 ) 史艳宇, 博士, 高级工程师, 主要研究方向为食品安全检测技术研究 shiyanyu219@163.com

Microsoft Word 王颖 _1_

Microsoft Word 王颖 _1_ 4 5 Vol. 4 No. 5 2013 10 Journal of Food Safety and Quality Oct., 2013 PCR 王颖 1*, 史艳宇 1, 刘金华 2, 吕航 1, 王莹 1 (1., 130022; 2., 130062) 摘要 : 目的方法 12S rrna, PCR, ; ; 50 mg/kg DNA DNA, 结果,, ( 0.1 μg/kg) 结论,,