巢式pcr和sybrgreenⅰ实时pcr检测转基因大豆-中国农学通报.pdfVIP

中国农学通报 2012,28(15):233-237 Chinese Agricultural Science Bulletin 巢式PCR和SYBRGreenⅠ实时PCR检测 转基因大豆方法的研究 彭新凯，宋涛平，谭 舸，陈 娜，胡朝晖，杨丽霞 （长沙市食品质量安全监督检测中心，长沙410013 ） 摘 要：为了建立转基因大豆检测技术，采用巢式PCR 和SYBR Green Ⅰ实时PCR 技术，检测转基因大豆 外源基因(CaMV35S、CP4EPSPS) 。结果表明，利用巢式PCR 可检测出1 ng/µL 转基因含量1%的大豆中 的CaMV35S 基因，而第一轮PCR 的检测限为100 ng/µL ；利用SYBR Green Ⅰ染料能结合双链DNA 的 特点，应用实时PCR 技术可检测到CaMV35S、CP4EPSPS 基因扩增所产生的信号，通过扩增产物的熔解 曲线能有效地区分特异性产物，CaMV35S 基因的检测限为0.1 ng/µL 。同时利用该方法对黄豆、炒黄豆、 豆干等样品进行检测，样品中未检出CaMV35S 基因成分。巢式PCR 方法明显提高了PCR 的检测限， SYBR Green Ⅰ实时荧光PCR 方法能有效、快速检测CaMV35S 转基因成分。 关键词：转基因大豆；巢式PCR ；SYBR Green Ⅰ实时PCR ；熔解曲线 中图分类号：Q78 文献标志码：A 论文编号：2012-0429 TheDetectionofExogenousGeneswithNestedPCRandSYBRGreenⅠRealTimePCR inGeneticallyModifiedSoybeanLine Peng Xinkai, Song Taoping, Tan Ke, Chen Na, Hu Zhaohui, Yang Lixia (ChangshaCenterofSupervisionInspectiononFoodQualitySafety, Changsha 410013) Abstract:Nested PCR and fluorescent SYBR Green Ⅰreal time PCR were developed for detection of several foreign genes (CaMV35S, CP4EPSPS) in genetically modified soybean. The results showed that, nested PCR could detect CaMV35S in 1 ng/µL DNA and the detection limit was 100 ng/µL in the first PCR. SYBR Green Ⅰreal time PCR could detect CaMV35S, CP4EPSPS genes and the detection limit was 0.1 ng/µL for CaMV35S. Different kinds of soybean foodstuffs were detected by nested PCR and real time PCR, the results showed that, no CaMV35S gene was detected in all the foodstuffs. Therefore, nested PCR and the SYBR Green Ⅰreal time PCR were the effective methods for detecting GM soybean foodstuffs. Keywords:roundup ready soybean (RRS); nested PCR; SYBR Green Ⅰreal-time PCR; melting curves