转基因玉米MIR162转化事件特异性检测方法及其标准化

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。

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

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.