番茄叶片基因组DNA快速制备技术及其在基于实时荧光定量PCR的转基因检测中的应用

Techniques for rapid preparation of tomato leaf DNA and its application in real-time quantitative PCR-based transgene detection

以番茄（Solanum lycopersicum L.cv.Micro-Tom）叶片为试材,建立了一种简便快速制备叶片基因组DNA的方法。2~20 mm2的叶片即可满足制备要求,制备过程只需一种提取试剂、只涉及1次移液和1次离心操作,不涉及沉淀。确定了所制备的DNA用于实时荧光定量PCR的合适用量为0.1~0.2μL（反应总体积为12.5μL）,发现过量模板的使用可降低PCR效率且可导致扩增失败。该项DNA快速制备及相适应的实时荧光定量PCR技术已成功应用于番茄转基因植株检测。

Using tomato（Solanum lycopersicum L.cv.Micro-Tom） leaf as material,a simple and rapid DNA preparation protocol was established.This method required only 2-20 mm2 leaf with only one extraction solution and involved one pipetation and one centrifugation each.No precipitation was required.The suitable volume of prepared DNA solution,as PCR template,for real-time quantitative PCR was determined to be 0.1-0.2 μL in 12.5 μL final reaction volume.The ex-cessive template DNA solution was confirmed to reduce PCR efficiency and even can result in PCR failure.This technique for rapid preparation of DNA and a compatible real-time quantitative PCR were successfully applied in transgene detection of tomato plants.