转基因延熟番茄“华番一号”的品系特异性检测方法

Event-specific transgenic detection of genetically modified Tomato Bioscein with real time PCR method.

本文使用反向PCR方法克隆了转基因延熟番茄“华番一号”(Bioscein)的外源基因和番茄基因组之间的一段边界序列,并依据此段序列设计了具有品系特异性的引物和荧光探针,以实时荧光PCR技术建立了华番一号的品系鉴定检测方法,扩增片段长108bp,横跨在Nos启动子和番茄基因组之间。以转基因大豆(RR)、转基因玉米(Mon810)、转基因抗草甘膦油菜、转基因棉花(保龄棉)、非转基因番茄、马铃薯、茄子、大椒、大米、小麦、烟草等为试材,证明本方法同其它转基因作物及其它蔬菜无非特异性反应。本方法在检测华番一号番茄时,相对检测灵敏度可达到0.1%,绝对灵敏度达到20个拷贝。由于本方法的PCR扩增产物长度只有108bp,因此该方法也可以用于检测加工产品中的转基因成分,或作为常规PCR定性检测后的确证实验方法。

We report the characterisation of the 5＇-flanking sequence of integrated gene-construct of the genetically modified Tomato （ Bioscein）, and the successive development and validation of a transgenic event-specific TaqMan 5＇-nuclease PCR assay for detection of Bioscein tomato on the ABI 7700 sequence detector. The limit of detection in pure tomato materials was estimated to approximately 20 template copies. Because of the low number of template copies required for detection, in combination with small size of the amplicon（ 108 bp）, the assay may be applied to analyses of raw materials as well as processed food.