转基因棉花MON88913转化体特异性定性、定量PCR检测方法

本文以我国批准商业化的转基因耐草甘膦棉花MON88913为研究对象,建立并验证了其转化体特异性定性、定量PCR检测方法.建立的定性PCR方法的检测极限是20个拷贝棉花单倍体基因组DNA,定量PCR方法的检测和定量极限分别是10和20个拷贝棉花单倍体基因组DNA.同时,我们组织了实验室5位研究人员对建立的定量PCR检测方法进行了协同验证.对5个盲样的定量分析结果显示与真实值的偏差介于1.59% 和10.12%之间,完全满足国际标准25%偏差范围的要求,完全可用于转基因棉花MON88913的实际样品检测.

Event-specific qualitative and quantitative PCR detection methods for genetically modified cotton MON88913

MON88913 is one glyphosate-tolerant genetically modified (GM) cotton event, and which has been approved for using as processed materials since 2007 in China. The aim of this study was to establish event-specific qualitative and quantitative detection methods for MON88913. In this study, the specific primers and TaqMan probes based on the revealed 3' end flanking sequence of GM cotton MON88913 exogenous integration were designed, and the qualitative and quantitative PCR assays were established employing the designed primers and probes. The qualitative PCR assay produced a 231-b Pproduct and the limit of detection (LOD) was 0.05% in 100 ng total cotton genomic DNA, corresponding to about 20 copies haploid cotton genomic DNA. The limit of detection and quantification (LOD and LOQ) of the established quantitative PCR assay were determined to be approximately 10 and 20 copies haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR method was in-house validated by 5 different researchers. In this validation, 5 mixed cotton samples with known MON88913 contents were quantified employing the developed quantitative PCR method, and the quantified bias between the true and quantification value from 5 researchers were ranged from 1.59% to 10.12%. All these results suggested that the developed qualitative and quantitative PCR methods were applicable for the identification and quantification of GM cotton MON88913 and its derivates.