巢式PCR和SYBR Green Ⅰ实时PCR检测转基因大豆方法的研究

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。

The Detection of Exogenous Genes with Nested PCR and SYBR Green Ⅰ Real Time PCR in Genetically Modified Soybean Line

Nested PCR and fluorescent SYBR Green Ⅰ real 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 Ⅰ real 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 Ⅰ real time PCR were the effective methods for detecting GM soybean foodstuffs.