【摘要】 为给转基因植物监测提供技术支持 ,建立了转基因“华番一号”番茄筛选和特异性的定性、定量PCR检测方法。转基因“华番一号”的筛选PCR检测主要以转基因通用元件CaMV35S启动子和NOS终止子为目的基因片段 ,特异性PCR检测以转基因外源重组子的CaMV35S启动子和反义EFE基因的相邻序列为目的片段 ;实验同时设立番茄的LAT5 2基因为转基因番茄定性、定量PCR检测的内对照基因。在所建立的PCR检测体系中 ,定性PCR筛选和特异性检测的检测极限为 6 8个拷贝 ,实时定量PCR方法的检测极限为 3个拷贝 ;筛选定量PCR检测的定量极限为 3个拷贝 ,特异性定量PCR检测的定量极限为 2 5个拷贝。最后通过对 2个已知含量的转基因番茄“华番一号”混合试样的检测 ,证明了该体系可以有效地用于转基因番茄“华番一号”的筛选和特异性的定性、定量PCR检测。更多还原

【Abstract】 Genetically modified (GM) tomatoes have been approved for commercialization in many countries since the first GM tomato FLAVR AVAR was permitted for planting in 1994. In China, GM tomato BIOSCIEN with a character of long shelf life was the first GM plant approved for commercialization in 1996. To meet the requirement of GM tomatoes labeling policy that has been actualized in China since 2001, the screening and construct specific PCR detection for detecting the universal elements transformed into tomato, such as Cauli flower mosaic virus 35 S(CaMV35S ) promoter and the nopaline synthase ( NOS ) terminator of Agrobacterium tumefaciens , and the specific inserted heterologous DNA sequence between CaMV35s promoter and anti sense ethylene forming enzyme (EFE ) gene were set up, respectively. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection (LOD) of screening and construct specific detection for BIOSCIEN was 68 haploid genome copies in conventional qualitative PCR detection, and 3 copies in TaqMan real time PCR detection. The limit of quantification (LOQ) of screening quantitative PCR assay for BIOSCIEN was 3 copies and 25 copies for construct specific quantitative PCR. Two samples with known BIOSCIEN tomato contents were detected using the established conventional and real time PCR systems,and the results indicated that the established BIOSCIEN screening and construct specific PCR detection systems were reliable, sensitive, and accurate.更多还原