Microsoft Word 王颖 _1_

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1 4 5 Vol. 4 No 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., ; 2., ) 摘要 : 目的方法 12S rrna, PCR, ; ; 50 mg/kg DNA DNA, 结果,, ( 0.1 μg/kg) 结论,, 关键词 : ; 12S rrna ; real time PCR; Detection of porcine-derived materials in meat products by real time PCR method WANG Ying 1*, SHI Yan-Yu 1, LIU Jin-Hua 2, Lv Hang 1, WANG Ying 1 (1. Jilin Product Quality Supervision Inspection, Changchun , China; 2. Jilin Entry-Exit Inspection and Quarant Bureau, Changchun , China) ABSTRACT: Objective To establish a rapid, specific, sensitive detection method for porcine derived materials. Methods A real-time PCR method for the porcine derived materials detection in meat products was established using forward and reverse primers and probe corresponding to the mitochondrial 12S rrna gene. The specific detection was carried out by livestock and poultry meat including lamb, beef, chicken, goose, duck, rabbit meat, horse meat, venison and other reference animal species and 50 mg/kg lamb DNA was chosen as diluent by gradient dilution of pork DNA, then sensitivity detection was executed. Results This method could effectively detect the porcine-derived materials, and showed a strong specificity and high sensitivity (up to 0.1 μg/kg), and the mutton components had no obvious influence on pork sensitivity detection. Conclusion This method could detect the porcine-derived components in meat products rapidly and accurately. KEY WORDS: porcine-derived materials; 12S rrna gene; real time PCR; detection,,, PCR PCR DNA [1-5],, GB/T 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 :,, jlszjywy@163.com *Corresponding author: WANG Ying, Master, Jilin Product Quality Supervision Inspection, No.1088, Dongnanhu Road, Nanguan District, Changchun , China. jlszjywy@163.com

2 PCR SN/T PCR, PCR,, PCR ; ( TaKaRa ),,, [6,7], 12S rrna gene,, 1 材料与方法 1.1 材料与试剂 ( ) ; AxyPrep TM DNA ( Axygen ); TaqMan Universal PCR Master Mix ( ABI ); K (20 mg/ml, ); CTAB( CTAB 20 g EDTA 7.4 g Tris 12.1 g 10% HCl, ph 8.0, ) Tris 70% 1.2 主要仪器 ( Beckman ); (IKA M20, IKA Labortechnik, Staufen, ); PCR (ABI 7500 ); DNA/RNA/ ( Bio Specmini ); (0.1~ μl Jilson ); 1.3 方法样品前处理,, : 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 12S rrna (accession AB ) Primer Express 3.0, GeneBank (http: // nih.gov/blast), Blast,, Forword primer: 5 - CAT GCG TAT CAC CAC CAT TAT ATC-3, Reverse primer: 5 - TGC CAA GCG GGT TGC T-3, Probe: FAM- CGA GCT TAA CTA CCA TGC CGC GTG A-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, 60 1 min, 特异性实验灵敏度实验 DNA(50 mg/kg) DNA 10

3 5, : PCR 1531, DNA µg/kg DNA, PCR, 荧光 PCR 方法用于实际样品检测, DNA, PCR 荧光 PCR 方法用于检测加工肉制品中猪源性成分,, DNA, PCR 2 结果与分析 2.1 引物特异性验证 PCR, DNA PCR, ( ),, 灵敏度实验 DNA DNA PCR,, 0.1 µg/kg, DNA 市售肉卷和肉板中的猪源性成分检测 4 ( ) PCR,, 市售加工肉制品中猪源性成分检测,, DNA PCR,, PCR, 4 Fig. 1 1 PCR Result of specificity test by real time PCR

4 1532 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 by real time PCR Curve from left to right: µg/kg; 10.0 µg/kg; 1.0 µg/kg; 0.1 µg/kg Fig. 3 图 3 检测样品中的猪源性成分 Determination of porcine-derived materials from sold products Curve from left to right: porcine rolls1 2 and porcine board 3 4. 第4卷

5 5, : PCR 1533 Fig. 4 4 Determination of porcine-derived materials from processed products 3 讨论 DNA PCR( ) DNA,, PCR,, [8-11],, GB/T PCR SN/T PCR [6,7], PCR,, PCR ;, ( TaKa- Ra ),,,,,,,,,, 12S rrna,,,,,,,, 50 mg/kg DNA DNA, DNA, 0.1 μg/kg,,, DNA, DNA,, [12-14]

1534 4,,, 参考文献 [1] SN/T 2557-2010 PCR [S]. SN/T 2557-2010 Detection of qualitation of bovine ingredient in meat product- Real time PCR method [S]. [2] SN/T 2727-2010 PCR [S].

6 1534 4,,, 参考文献 [1] SN/T PCR [S]. SN/T Detection of qualitation of bovine ingredient in meat product- Real time PCR method [S]. [2] SN/T PCR [S]. SN/T Deteumination of poultry derived materials in feedstuff-real-time fluorescent PCR method [S]. [3] GB/T PCR [S]. GB/T Identification of cervus derived materials in animal-originated feedsatuffs product-pcr method [S]. [4] GB/T ( ) PCR [S]. GB/T Identification of Ruminantia derived materials (bovidia caprinae cervus) in animal-originated feedsatuffs -PCR method [S]. [5] GB/T PCR [S]. GB/T Identification of Canis derived materials in animal-originated feedsatuffs product-pcr method [S]. [6] SN/T PCR [S]. SN/T Determination of bovine, ovine, porcine derived materials in food, cosmetic and feed-real time PCR method [S]. [7] GB/T PCR [S]. GB/T Identification of Porcine derived materials in animal-originated feedsatuffs-pcr method [S]. [8],,,. [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. [9] Yang BH, Zong H. Determination of animal derived materials with conserved mitochondrial sequence in feedstuffs [J]. Feed Res, 2002, 5: 1 3. [10],,,. [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. [11] 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): [12] 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: [13] 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): [14] 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 : 周盼虹 ) 王颖, 硕士, 主要研究方向为食品生物技术 jlszjywy@163.com

Microsoft Word 史艳宇-确定版面.doc

Microsoft Word 史艳宇-确定版面.doc 4 6 Vol. 4 No. 6 2013 12 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., 130022; 2., 130062; 3., 132600) 1 摘要 : 目的方法, PCR, ; ; 50 mg/kg