转基因苜蓿草J101品系特异性实时荧光PCR检测方法的建立

Event-specific Real-time PCR detection of genetically modified alfalfa events J101

本文针对转基因苜蓿草品系J101,设计引物及探针建立J101品系特异实时定量PCR检测体系。结果表明,本文建立的定量实时PCR检测方法特异性良好。实时荧光定量PCR检测方法的检测下限为10拷贝J101基因组DNA,定量检测下限为50拷贝的J101基因组DNA,本方法建立的标准曲线线性相关系数(R2)为0.993,扩增效率E为110%,重复性实验显示本方法的标准偏差(SD)及相对标准偏差(RSD)都在可接受范围内。本文建立的J101品系特异性定量PCR检测方法适于快速、准确、稳定地对转基因苜蓿草J101品系进行检测。

To establish event-specific quantitative TaqMan Real-time PCR detection methods for genetical- ly modified（GM） alfalfa events J101,the specific primer pairs and probe based on the 5＇junction sequence spanning the alfalfa genomic DNA and transgene insert DNA of J101 were designed. Results showed that the developed quantitative realtime PCR detection method were specific for J101 detection. The limit of detection （LOD）and limit of quantification （LOQ） of quantitative Real-time PCR method were 10 and 50 copies of J101 genomic DNA,respectively. The correlation coefficient of standard curve were 0.993 and the efficiency of Real-time PCR was 110%.Repeat ability of the established event-specific Real-time PCR method was assessed and the standard deviation （SD） and the relative standard deviation（RSD） were all in the acceptable range. Therefore, the established event-specific quantitative Real-time PCR method suit for GM alfalfa J101 samples detection quickly and accurately.