口岸重点监控的转基因棉花T304-40品系定量PCR检测方法

Quantitative PCR detection of genetically modified cotton T304-40 monitored emphatically at ports

以我国口岸重点监控的转基因棉花T304-40品系为研究对象,根据其外源插入片段与基因组DNA3'端邻接区序列设计引物和探针,经筛选和优化后,建立了转基因棉花T304-40品系特异性实时荧光定量PCR检测方法。对方法的适用性测试结果表明,建立的实时荧光定量PCR检测方法高度特异于转基因棉花T304-40品系检测,反应效率达94.2%,绝对灵敏度为20拷贝基因组DNA,定量下限为25拷贝基因组DNA。以研制的检测方法建立标准曲线对盲样进行定量分析,结果表明4个盲样的测定值与设定值间的偏差均小于10%。因此,本研究建立的转基因棉花T304-40品系特异性实时荧光定量PCR方法特异性强、灵敏度高,可准确地对棉花及其制品中转基因棉花T304-40成分进行定性或定量检测分析。

In order to identify and detect genetically modified（GM）cotton T304-40,which was monitored emphatically at Chinese ports,primers and probes were designed according to the 3＇ flanking sequences of the foreign insert and cotton genomic DNA,and via screening and optimization,an event-specific fluorescent quantitative real-time PCR method to detect GM cotton T304-40 was established.Tests for the method applicability showed that the established real-time PCR method was highly specific to GM cotton T304-40 detection,the reaction efficiency was 94.2%,and the absolute detection and quantification limits were 20 and 25 copies of genomic DNA respectively.The quantitative analysis of four blind samples by using the established realtime PCR method showed that the deviations between the measured values and the set values were all less than10%.All the results demonstrated that the established real-time PCR method to detect GM cotton T304-40 has high specificity and sensibility and can be applied to qualitative and quantitative analyses of T304-40 ingredient in cotton and its derived products.