欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2013, Vol. 39 ›› Issue (07): 1141-1147.doi: 10.3724/SP.J.1006.2013.01141

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

转基因玉米MIR162转化事件特异性检测方法及其标准化

张广远1,2,孙红炜1,李凡1,杨淑珂1,路兴波1,*,赵蕾2,*   

  1. 1山东省农业科学院植物保护研究所，山东省植物病毒学重点实验室，山东济南250100；2山东师范大学生命科学学院，山东济南250014
  • 收稿日期:2012-12-27 修回日期:2013-02-15 出版日期:2013-07-12 网络出版日期:2013-04-23
  • 通讯作者: 路兴波, E-mail: luxb99@sina.com, Tel: 0531-83179095; 赵蕾, E-mail: zhaolei@sdu.edu.cn, Tel: 0531-88177190
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2011ZX08012-004)资助。

Event-Specific PCR Detection Method of Genetically Modified Maize MIR162 and Its Standardization

ZHANG Guang-Yuan1,2,SUN Hong-Wei1,LI Fan1,YANG Shu-Ke1,LU Xing-Bo1,*,ZHAO Lei2,*   

  1. 1 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Plant Virology, Jinan 250100, China; 2 College of Life Science, Shandong Normal University, Jinan 250014, China
  • Received:2012-12-27 Revised:2013-02-15 Published:2013-07-12 Published online:2013-04-23
  • Contact: 路兴波, E-mail: luxb99@sina.com, Tel: 0531-83179095; 赵蕾, E-mail: zhaolei@sdu.edu.cn, Tel: 0531-88177190

RichHTML

PDF

2204

被引次数

9

摘要：

转基因玉米MIR162是美国先正达公司开发出来的新型抗鳞翅目昆虫的转基因玉米品种。本研究目的在于建立该品系转化事件特异性定性、定量检测方法并形成定性检测国家标准。定性PCR体系经实验室内部比对及实验室间循环验证结果表明，该体系特异性强，灵敏度较高(0.1%)，具有良好的重复性、再现性。Taqman探针实时荧光PCR检测结果表明，所设计的探针特异性强，检测低限(LOD)在0.01%以下；定量标准曲线R2均为0.999，扩增效率为98.622%和99.602%，SD范围为0.014~0.209，RSD的范围为0.049%~0.879%，稳定性好；对转基因含量为1.0%、0.5%、0.1%的样品定量结果相对偏差在8%以内，结果较为准确。本研究建立的方法完全可用于含转基因玉米MIR162成分样品的检测。

关键词: 转化体特异性, MIR162, 定性PCR, Taqman探针实时荧光PCR, 标准化

Abstract:

Genetically modified maize MIR162 with resistances to Lepidoptera insects is developed by U.S. Syngenta Company through recombinant DNA technology. The purpose of this study was to establish and standardize the event-specific detection method of this line in China. The primers and Taqman probe were designed based upon the revealed 3' and 5' flanking sequences of MIR162. After validated by six external laboratories, results showed that the qualitative PCR detection method could specifically detect the samples of MIR162 with the detection sensitivity about 0.1%, a good repeatability and reproducibility. The quantitative PCR system was based on TaqMan. Two standard curves and corresponding linear regression equations of Quantitation-Ct between endogenous zSSIIb gene and transgenic target gene of MIR162 maize in standard material were generated. Results showed that the standard curves had good linear relationships. Their R2 values both were 0.999 and the recovery rates were 98.622% and 99.602%. Moreover, the standard deviations (SD) and relative standard deviations (RSD) of repeatability ranged from 0.014 to 0.209 and 0.049% to 0.879%. The limit of detection (LOD) was 0.01%. Thereafter, three mixed corn samples containing 1.0%, 0.5%, and 0.1% MIR162 were quantified employing the developed quantitative PCR method, and the quantified bias between the true value and tested value was below 8%. All these results suggested that the developed qualitative and quantitative PCR methods can be used for the identification and quantification of GM maize MIR162.

Key words: Event-speci?c, MIR162, Qualitative PCR, Taqman real-time PCR, Standardization

[1]Wei J-J(魏俊杰). Detection methods of transgenic products. J Hebei Agric Sci (河北农业科学), 2011, 15(11): 48–49 (in Chinese with English abstract)



[2]Forte V T, Di-Pinto A, Martino C, Tantillo G M, Grasso G, Schena F P. A general multiplex-PCR assay for the general detection of genetically modified sya and maize. Food Control, 2005, 16: 535–539



[3]Xu W-T(许文涛), Bai W-B(白卫滨), Luo Y-B(罗云波), Yuan Y-F(元延芳), Huang K-L(黄昆仑). Research progress in detection technique for genetically modified organisms. J Agric Biotechnol (农业生物技术学报), 2008, 16(4): 714–722 (in Chinese with English abstract)



[4]Gachet E, Martin G G, Vigneau F, Meyer G. Detection of genetically modified organism (GMOs) by PCR: A brief review of methodologies available. Trends Food Sci Technol, 1999, 9: 380–388



[5]Isabel T, Pieter W, Marc V, Anne M, Erik V B, Guy V E, Marc D L. Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176 and GA21 maize and transgenic GT73 canola. J Agric Food Chem, 2005, 53: 3041–3052



[6]Yang L T, Pan A H, Zhang K W, Yin C S, Qian B J, Chen J X, Huang C, Zhang D B. Qualitative and quantitative PCR methods for event-speci?c detection of genetically modi?ed cotton Mon1445 and Mon531. Transgenic Res, 2005, 14: 817–831



[7]Wu G, Wu Y H, Xiao L, Lu C M. Event-specific qualitative and quantitative PCR methods for the detection of genetically modified rapeseed Oxy-235. Transgenic Res, 2008, 17: 851–862



[8]Pan A H, Yang L T, Xu S C, Yin C S, Zhang K W, Wang Z Y, Zhang D B. Event-speci?c qualitative and quantitative PCR detection of MON863 maize based upon the 3'-transgene integration sequence. J Cereal Sci, 2006, 43: 250–257



[9]Yang L T, Xu S C, Pan A H, Yin C S, Zhang K W, Wang Z Y, Zhou Z G, Zhang D B. Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence. J Agric Food Chem, 2005, 53: 9312–9318



[10]Katarina C, Valérie C A, Marie-Noelle F, Kristina G, André K, Jana ?, Yves B. Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize. Anal Biochem, 2008, 376: 189–199



[11]Yang L T, Guo J C, Pan A H, Zhang H B, Zhang K W, Wang Z M, Zhang D B. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J Agric Food Chem, 2007, 55: 15–24



[12]Sissel B R, Marc V, Knut G B, Arne H J. Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays). Eur Food Res Technol, 2003, 216: 347-354



[13]Hari K S, Kae-Kang H, Wang S J, Liu L F, Chang M C. Simultaneous detection of eight genetically modified maize lines using a combination of event and construct specific Multiplex-PCR technique. J Agric Food Chem, 2008, 56: 8962–8968



[14]Tigst D, Indira R. Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73 canola event. Food Control, 2008, 19: 893–897



[15]Li X, Shen K L, Yang L T, Wang S, Pan L W, Zhang D B. Applicability of a novel reference molecule suitable for event-specific detections of maize NK603 based on both 5' and 3' flanking sequences. Food Control, 2010, 21: 927–934



[16]Rott M E, Lawrence T S, Wall E M. Development of real-time PCR systems based on SYBR Green I amplifluore and Taqman technologies for specific quantitative detection of of the transgenic maize event GA21. Cereal Sci, 2004, 39: 99–107



[17]Jiang G-Z(姜国忠), Xie H(谢华), Guo Y-Z(郭玉忠), Fan T-L(范天黎), Xue L-X(薛乐勋). The comparison between two techniques in isolating 3' flanking region of Dunaliella salina actin gene. J Zhengzhou Univ (Med Sci)(郑州大学学报?医学版), 2004, 39(1): 41–44 (in Chinese with English abstract)
[1] 李鹏,张琳,叶吉妮,贺诗瑶,贾军伟,潘爱虎,唐雪明. 抗病转基因水稻M12及其产品成分的定性、定量PCR检测方法[J]. 作物学报, 2018, 44(7): 949-955.
[2] 李俊，刘信，曹应龙，武玉花，厉建萌，吴刚，张丽，卢长明. 植酸酶基因定性PCR检测方法及阳性质粒分子的构建[J]. 作物学报, 2012, 38(04): 639-647.
[3] 袁磊，孙红炜，赵蕾，杨崇良，尚佑芬，路兴波. 转基因玉米MON88017旁侧序列分析及定性PCR检测[J]. 作物学报, 2010, 36(2): 361-364.
[4] 路兴波,武海斌,王敏,李宝笃,杨崇良,孙红炜. 转基因玉米转化体特异性寡核苷酸芯片测试方法的研制[J]. 作物学报, 2009, 35(8): 1432-1438.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2