Microsoft Word 王清华校对稿

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1 4 3 Vol. 4 No Journal of Food Safety and Quality Jun., 2013 BT11 (LAMP) 王清华 1, 徐君怡 2, 曹冬梅 2, 刘宇 2, 曹际娟 (1., ; 2., ) 2* 摘要 : 目的 BT 11 (LAMP) 方法 BT 11 (AY AY629236), LAMP, 结果, BT 11 ; 0.5%;, 结论 LAMP BT 11, 关键词 : BT 11; (LAMP); Establishing a loop-mediated isothermal amplification(lamp)detection method for genetically modified maize BT11 WANG Qing-Hua 1, XU Jun-Yi 2, CAO Dong-Mei 2, LIU Yu 2, CAO Ji-Juan 2* (1. School of Biological Engineering, Dalian Polytechnic University, Dalian , China; 2. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian , China) ABSTRACT: Objective To establish a loop-mediated isothermal amplification(lamp)detection method for BT 11. Methods The primers were designed according to the specific sequence(ay , AY629236)of BT 11, and then the LAMP reaction system was established. The specificity, sensitivity and stability of the system were analyzed. Results The primers in this research specifically detected the BT 11. The detection sensitivity was up to 0.5%, and the precision of the experimental analysis was proved to be of high stability. Conclusion The results showed that LAMP was a specific, stable, rapid, sensitive and visible way for the detection of BT 11, and it would have broad prospects for application. KEY WORDS: genetically modified maize BT 11; LAMP; identification of lines 1 引言, [1],, BT 11, [2],,, 基金项目 : (2013ZX ) Fund: Supported by Major Program of National Genetically Modified Foundation of China (2013ZX ) * 通讯作者 :,, cjj0909@163.com *Corresponding author: CAO Ji-Juan, Professor, Liaoning Entry-Exit Inspection and Quarantine Bureau, No. 60, Changjiang East Road, Dalian , China. cjj0909@163.com

2 3, : BT11 (LAMP) 869, [3], (PCR) 2000, Notomi [4] DNA (loop-mediated isothermal amplification of DNA, LAMP) LAMP, DNA 6 4 Bst DNA,, [5] LAMP, [6] 2 材料与方法 2.1 实验材料 BT 11 (ERMBF 412F, sigma) BT 176 (ERMBF 411F, sigma) GA 21 (0407-B, AOCS) MON 810 (ERMBF 413EK, sigma) MIR 604 (0607-A2, AOCS) MON (0406-D, AOCS) MON (0906-E, AOCS) EVENT MON 863; BT ; EH (0806-C, AOCS); A (0707-C3, AOCS) GTS DP DP MON 89788; GT 73(0304-B, AOCS);, 2.2 基因组 DNA 的提取 2.1,, DNA Extraction Kit for GMO Detection Ver.2.0 (TaKaRa Code No.D9093) DNA DNA 100 µl TE, (NanoDrop) DNA OD 260 /OD 280 DNA, DNA OD LAMP 引物设计 [7 10], NCBI BT 11 (AY AY629236), Primer Explorer version 4( jp/e/) LAMP Designer LAMP 2 2, 6 FIP F1c( Fl ) F2, BIP B1c( B1 ) B2 ; F3 B3 2 FLP BLP, LAMP 反应体系的建立 25 µl LAMP, : 1 ThermoPol F3 B3 0.2 µmol/l FIP BIP 1.6 µmol/l 1.4 mmol/l dntps 0.8 mmol/l 6 mmol/l 0.32 U/µL Bst DNA DNA 200 ng, 23 µl, Table 1 表 1 转基因玉米 BT11 品系的 LAMP 引物 LAMP primers for genetically modified maize BT11 F3 B3 FIP(F1c+F2) BIP(B1c+B2) FLP BLP (5 3 ) GCTGTAGCTGGCCTAATC GGCCAAGGTATCTAATCAGC TATCTGTCTCAGGGGCAGACTCTCAACTGGTCTCCTCTCC GCCAAGAAGGCGCAAGTCCATCCCATTTGTGATCTTTGTC GTGTTCCCTCGGATCTCG ACCGCGAGTTGTTGTATCATA

3 870 食品安全质量检测学报 20 μl 密封液, 再分别加入 2 µl 模板 DNA 或阴阳性对照模板, 混匀离心后将 1 µl 显色液点在管盖中应的特异性, 并利用 LAMP 浊度仪观察反应结果 2.6 间, 小心盖紧管盖, 避免显色液掉入反应液中参照 LAMP 浊度仪使用说明书, 将混合物置于反应孔中, 65 恒温反应 60 min, 最后 80 下灭活 5 min 结束反应 2.5 第4卷 LAMP 检测灵敏度实验将含量为 5%的转基因玉米 BT 11 品系 DNA(100 ng/μl)用非转基因玉米 DNA(100 ng/μl)分别稀释至转基因成分含量为 2% 1% 0.5% 0.1% 的 4 个梯度, 每个稀释度分别取 2 µl 进行 LAMP 实验, 用 LAMP 引物特异性实验 ddh2o 作为阴性对照, 转基因玉米 BT 11 品系用转基因玉米 BT 11 品系 DNA 和豌豆扁豆 DNA(5%, 100 ng/μl)作为阳性对照, 表 1 中的引物作大米羽扇豆土豆玉米大豆螃蟹绿豆核为 LAMP 引物, 进行 LAMP 反应, 63 下运行 60 桃胡萝卜小麦转基因水稻 BT 63 科丰 6 号 min, 以确定 LAMP 检测的灵敏度, 并根据最低灵敏科丰 8 号转基因玉米 BT 176 EVENT GA 度的出峰时间及假阳性的最早出峰时间, 确定反应 21 MON 863 MON 810 MIR 604 MON 时间 MON 转基因土豆 EH 转基因大豆 A GTS DP DP MON 转基因油菜 GT 73 的 DNA 作为 LAMP 反应模板, 表 1 中的引物作为 LAMP 引物, 进行 LAMP 反应, 63 下运行 60 min, 以验证 LAMP 反 Fig 结果 LAMP 引物特异性根据 2.5 中 LAMP 引物特异性实验的方案, 进行 LAMP 反应, 结果如图 1 所示图 1 LAMP 引物特异性检测图 The LAMP primers specific detection figure 注: 图 1A. CH1: 阳性对照(BT 11), CH2: 阴性对照(超纯水), CH3: DP , CH4: RT 73, CH5: MON 89034, CH6: MON 89788, CH7: MON 88017, CH8: MON 863; 图 1B. CH1: MIR 604, CH2: A , CH3: DP , CH4: GTS , CH5: EVENT 98140, CH6: BT 176, CH7: MON 810, CH8: GA 21; 图 1C. CH1: BT 63, CH2: 科丰 6 号, CH3: 科丰 8 号, CH4: EH , CH5: 绿豆, CH6: 核桃, CH7: 胡萝卜, CH8: 小麦; 图 1D. CH1: 豌豆, CH2: 扁豆, CH3: 大米, CH4: 羽扇豆, CH5: 土豆, CH6: 玉米, CH7: 大豆, CH8: 螃蟹

4 第3期王清华, 等: 转基因玉米 BT11 品系环介导等温扩增(LAMP)检测方法的建立 871 由图 1 可见, 除图 1A 中的 CH1(转基因玉米 BT 11 品系 DNA)外, 其余 30 种样品均未出现阳性峰这说明本研究设计的引物只对转基因玉米 BT 11 品系有特异性检出, 且特异性良好 3.2 LAMP 检测灵敏度根据 2.6 中 LAMP 检测灵敏度实验的方案, 进行 LAMP 反应, 结果如图 2 所示 Fig. 2 图 2 LAMP 引物检测的灵敏度 The sensitivity of the LAMP detection 注: CH1: 5% BT 11 标准样品; CH2: 2% BT 11 模拟样品; CH3: 1% BT 11 模拟样品; CH4: 0.5% BT 11 模拟样品; CH5: 0.1% BT 11 模拟样品; CH6: 阴性对照(超纯水) 由图 2 可见, 不同浓度的转基因玉米 BT 11 品系 DNA 进行 LAMP 扩增时, 出峰时间和浓度呈明显的梯度变化, 检测灵敏度可达 0.5%; 此外, 最低检出限 0.5%约在 36 min 出峰, 且未出现假阳性结果, 故可将 LAMP 反应的时间确定为 60 min 3.3 方法的稳定性将转基因玉米 BT 11 品系 DNA(5%, 100 ng/μl) 用非转基因玉米 DNA 稀释至 0.5%作为模板, 取 20 份模板作为平行样品, 以 ddh2o 作为阴性对照, 转基因玉米 BT 11 品系 DNA(5%, 100 ng/μl)作为阳性对照表 1 中的引物作为 LAMP 引物, 进行 LAMP 反应, 63 下运行 60 min, 结果如图 3 所示图 3 0.5%转基因玉米 BT11 品系 DNA 的精密度实验图 Fig % BT11 DNA precision experimental figure 由图 3 可见, 20 份 0.5%转基因玉米 BT 11 品系注: 图 3A. CH1: 阳性对照(5% BT11 DNA), CH2: 阴性对照(超纯 DNA 平行样品的平均出峰时间前后偏差均在 5 min 水), CH3-8: 0.5% BT11 DNA; 图 3B. CH1-8: 0.5% BT11 DNA; 图 3C. CH1-6: 0.5% BT11 DNA 内结果表明, 本方法检测精密度的稳定性良好 4 结论目前, 转基因玉米成分的检测方法主要为 TaqMan 实时荧光 PCR 检测法和普通 PCR 检测法普通 PCR 检测法由于操作繁琐污染环境灵敏度低等不足已被 TaqMan 实时荧光 PCR 检测法取代但是, TaqMan

872 4 PCR,, LAMP 4, PCR, Bst DNA 60, [11] LAMP,, DNA SYBR Green I,, ;, SYBR Green I, LAMP, LAMP [12-14] BT 11 LAMP,, PCR, 参考文献 [1], PCR MON 810 [J]., 2003, 11(1): 19-21 Cao JJ, Cao YY.

Quantitative/identified detection of the genetically modified maize BT11 components in processed products [J]. Biotechnol Bull, 2003, 6: 46 50. [3],,,.

5 872 4 PCR,, LAMP 4, PCR, Bst DNA 60, [11] LAMP,, DNA SYBR Green I,, ;, SYBR Green I, LAMP, LAMP [12-14] BT 11 LAMP,, PCR, 参考文献 [1], PCR MON 810 [J]., 2003, 11(1): Cao JJ, Cao YY. PCR on detection and identification of GM maize MON 810 [J]. Maize Sci, 2003, 11(1): [2],,. BT 11 PCR ( ) [J], 2003, 6: Qin W, Cao JJ, Zhu SF. Quantitative/identified detection of the genetically modified maize BT11 components in processed products [J]. Biotechnol Bull, 2003, 6: [3],,,. [J], 2010, 38(012): Yan XY, Jin XT, Wang Q, et al. The biosecurity of genetically modified maize [J]. Shanxi Agr Sci, 2010, 38(012): [4] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Res, 2000, 28(12): e63 e63. [5], 垚,,. LAMP [J]., 2010, 37(007): Jiang YH, Liu Y, Tan GL, et al. LAMP technology in food safety detection [J]. Guangdong Agr Sci, 2010, 37(007): [6], LAMP [J], 2004, 14(4): Wu YS, Luo SP. A novel high efficiency, rapid, isothermal nucleic amplification-lamp method [J]. Biotechnol, 2004, 14(4): [7],,, LAMP [J], 2010, 25(004): Lan QK, Wang Y, Zhao X, et al. Development and application of loop-mediated isothermal amplification for detection of GM maize [J]. Acta Agriculturae Boreali-Sinica, 2010, 25(004): [8],,, MON 863 [J], 2011, 19(1): Lan QK, Wang Y, Zhao X, et al. Study on rapid detection of transgenic maize MON 863 by LAMP [J]. Maize Sci, 2011, 19(1): [9],,, MON 89034[J], 2012, 28(4): Zhang J, Li ZY, Ye YX, et al. A specific loop-mediated isothermal amplification method for detection of transgenic maize MON [J]. Mod Food Sci Technol, 2012, 28(4): [10],,,. BioSunLAMP: [J]., 2012, 36(3): Cha L, Cai X, Ying XM, et al. BioSunLAMP: A software system for loop-mediated isothermal amplification (LAMP) primer design [J]. Mil Med Sci, 2012, 36(3): [11],,, LAMP [J], 2007, 27(12): Xu Q, Sun XH, Zhao Y, et al. Development of loop-mediated isothermal amplification ( LAMP ) method for detection of vibrio parahaemolyticus [J]. China Biotechnol, 2007, 27(12): [12] Dukes JP, King DP, Alexandersen S. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus [J]. Arch Virol, 2006, 151(6): [13] Mori Y, Kitao M, Tomita N, et al. Real-time turbidimetry of LAMP reaction for quantifying template DNA [J]. Biochem Biophys meth, 2004, 59(2): [14] Parida M, Posadas G, Inoue S, et al. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile Virus [J]. Clin Microbiol, 2004, 42(1): 作者简介 ( 责任编辑 : 佟丽 ) 王清华, 硕士, 主要研究方向为微生物学与分子生物学 wqhnana@qq.com 曹际娟, 研究员, 主要研究方向为食品安全检测与研究 cjj0909@163.com

Microsoft Word 聂雪梅.doc

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