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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

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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

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