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 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 130022, China; 2. Jilin Entry-Exit Inspection and Quarantine Bureau, Changchun 130062, China; 3. Jilin agricultural radio and Television School, Shulan 132600, 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 : (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: shiyanyu219@163.com *Corresponding author: SHI Yan-Yu, Associate Professor, Jilin Product Quality Supervision Inspection, No.1088, Dongnanhu Road, Nanguan District, Changchun 130022, China. E mail: shiyanyu219@163.com
1860 4 1 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, ); (0.1~1000.0 μl JILSON, ); 2.3 方法 [2] 2.3.1 样品前处理,, : 500 mg 50 ml, 2,, 12 000 r/min 5 min, 2,, 12 000 r/min 5 min, 1,, 2 ml, 12 000 r/min 5 min,, 12 000 r/min 5 min, 1 2.3.2 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), 12 000 r/min 15 min;, 12 000 r/min 10 min, ; 70% 2~3, ; 200 μl TE (TE DNA ); - (24:1, v/v) DNA DNA 2.3.3 引物设计 GenBank (accession KF156760) Primer Express 3.0, GenBank(http://www.ncbi.nlm.nih.gov/ 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-3 2.3.4 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 10 s, 58 32 s, 40 2.3.5 特异性实验 2.3.6 灵敏度实验 DNA(50 mg/kg) DNA 10, DNA 100.0 10.0 1.0 0.1 µg/kg DNA, PCR,
6, : PCR 1861 2.3.7 荧光 PCR 方法用于实际食品样品检测, DNA, PCR 2.3.8 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,,,,, 1 3.2 灵敏度实验 DNA DNA PCR, : 0.1 µg/kg, DNA 2 3.3 肉卷和肉板中的鸭源性成分检测 4 PCR, 3 3.4 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
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
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],,,
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