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,, 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 130022, China; 2. Jilin Entry-Exit Inspection and Quarant Bureau, Changchun 130062, 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 21101-2007 基金项目 : (QK20120169) Fund: Supported by the General Administration of Quality Supervision, Inspection and quarantine of the People s of Republic of China (QK20120169) * 通讯作者 :,, E-mail: jlszjywy@163.com *Corresponding author: WANG Ying, Master, Jilin Product Quality Supervision Inspection, No.1088, Dongnanhu Road, Nanguan District, Changchun 130022, China. E-mail: jlszjywy@163.com
1530 4 PCR SN/T 2051-2008 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 ); (0.1~1000.0 μl Jilson ); 1.3 方法 1.3.1 样品前处理,, : 500 mg 50 ml, 2,, 12000 r/min 5 min, 2,, 12000 r/min 5 min, 1,, 2 ml, 12000 r/min 5 min,, 12000 r/min 5 min, 1 1.3.2 DNA 的提取 DNA, DNA CTAB DNA: 200 mg 1.5 ml, 600 μl CTAB, 15 μl K, 65 30 min; 500 μl - - (25:24:1, v/v), 12000 r/min 15 min;, 12000 r/min 10 min, ; 70% 2 ~ 3, ; 200 μl TE (TE DNA ); - (24:1, v/v) DNA DNA 1.3.3 引物设计 GenBank 12S rrna (accession AB292606.1) Primer Express 3.0, GeneBank (http: //www.ncbi.nlm. 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 1.3.4 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, 95 15 s, 60 1 min, 40 1.3.5 特异性实验 1.3.6 灵敏度实验 DNA(50 mg/kg) DNA 10
5, : PCR 1531, DNA 100.0 10.0 1.0 0.1 µg/kg DNA, PCR, 1.3.7 荧光 PCR 方法用于实际样品检测, DNA, PCR 1.3.8 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, ( ),, 1 2.2 灵敏度实验 DNA DNA PCR,, 0.1 µg/kg, DNA 2 2.3 市售肉卷和肉板中的猪源性成分检测 4 ( ) PCR,, 3 2.4 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
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: 100.0 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, : PCR 1533 Fig. 4 4 Determination of porcine-derived materials from processed products 3 讨论 DNA PCR( ) DNA,, PCR,, [8-11],, GB/T 21101-2007 PCR SN/T 2051-2008 PCR [6,7], PCR,, PCR ;, ( TaKa- Ra ),,,,,,,,,, 12S rrna,,,,,,,, 50 mg/kg DNA DNA, DNA, 0.1 μg/kg,,, DNA, DNA,, [12-14]
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