多重PCR结合变性高效液相色谱技术转基因小麦检测方法的建立

Establishment of Multiplex PCR and DHPLC Method for Detecting GMO Wheat

为了满足对小麦转基因成分高通量快速检测的需要,建立了一种新的转基因小麦检测方法。通过对不同稀释倍数的转基因小麦品系B73-6-1中的内源基因Wx012、外源基因ubiquitin、bar、nos、uidA进行单一PCR和多重PCR扩增,应用琼脂糖凝胶和变性高效液相色谱(DHPLC,denaturing high performance liq-uid chromatography)技术分离PCR产物,进行灵敏度分析,并用非转基因小麦京花1号籽粒、大豆籽粒、麦片、转基因小麦B73-6-1加工成的油炸制品为样本,对建立的检测体系进行检测。结果表明,样品经多重PCR扩增和DHPLC分离后,能够得到准确可靠的转基因图谱。与凝胶电泳方法比较,本研究所建立的多重PCR-DHPLC具有更高的检测通量和灵敏度(能够同时检测5个基因,灵敏度达到1 ng.μL-1),能够满足小麦转基因成分的高通量快速检测的要求,同时也为转基因产品检测提供了一种新的技术手段。

This research was to set up a new method which was fast and high throughput for GMO wheat detection.Ubiquitin promoter,nos terminator,uidA（gus gene）,bar gene and the endogenesis Wx012 gene of various grads of GMO line B73-6-1 were amplified by single PCR（polymerase chain reaction）and multiplex PCR.These PCR products were separated by gel electrophoresis and DHPLC（denaturing high performance liquid chromatography）to test their sensitivity.Non GMO wheat Jing hua No.1,soya,wheat piece,fried food of GMO wheat line B73-6-1 were used to validate the application of this detection system.Results showed that the method of multiplex PCR and DHPLC was accurate,high throughput and got expected effection.Five genes was detected simultaneously.The detecting limitation of DHPLC was 1 ng·μL-1,which was better than gel electrophoresis.Multiplex PCR and DHPLC,as a new GMO detecting technique,can be used to detect transgenic modified components in wheat and other GMO products.