Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several realtime PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM) was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR wasfurther validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.%在很多国家,转基因生物(GMOs)及其衍生产品必须标有精确的转基因含量.最近研究中,实时定量PCR技术广泛应用于转基因成分的检测.然而,转基因生物的实时定量PCR方法的精确度仍然是一个难以解决的问题,尤其是对于高温处理过的样品.为了更好地准确定量高温处理样品中转基因的含量,对普通的实时定量PCR体系做了一些改进,包括重新设计内源基因和外源基因的引物,使得扩增较短并且大小接近的目标DNA片段,同时引物的GC含量和溶解温度也都相近.此外,采用热处理加工模型(HTPM)的方法,制备了含有转基因大豆GTS 40-3-2的样品,并验证了改进后的实时定量PCR系统.实验结果表明:使用改进后的实时定量PCR体系测定热处理过的样品,发现其中的转基因含量的定量偏差明显降低.同时使用改进的双重实时定量PCR进一步验证转基因大豆的加工食品,结果也显示,转基因含量的定量结果更准确.这些结果表明:改进的双重实时定量PCR将适用于热加工产品的定量检测.
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