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目的建立转Bt基因大米的多重降落PCR定性检测方法。方法本实验以转Bt基因大米为研究对象,针对大米的内源基因蔗糖磷酸合酶sps基因和转基因大米中普遍存在的外源基因CaMV35S启动子选择两对特异性引物,分别用于判断大米基因组DNA的存在及扩增体系的适用性和筛选转基因片段的存在,进行多重降落PCR扩增。分别对热循环参数和反应体系进行优化,同时考察了方法的特异性,并对市场出售的实际样品进行检测。结果在优化的条件下,转Bt基因大米的sps基因和CaMV35S启动子均可获得有效扩增。特异性实验结果表明本法特异性高,结果稳定;所选择的市售样品均未检测出转基因成分。结论本研究建立的转基因大米的多重降落PCR检测方法与常规PCR相比,更为快速、简便,且具有较强的特异性,可用于转基因大米的快速定性检测。
Objective To establish a qualitative multiplex PCR detection method for transgenic Bt rice. Methods In this study, Bt transgenic rice was selected as the research object. Two pairs of specific primers were selected according to the sps gene of rice endogenous gene sucMV35S and the common exogenous gene CaMV35S in transgenic rice, respectively, to determine the rice genome DNA presence and the applicability of the amplification system and the presence of transgene fragments were screened for multiple fall PCR amplification. The thermal cycling parameters and the reaction system were optimized respectively, the specificity of the method was also examined, and the actual samples sold in the market were tested. Results Under the optimized conditions, the sps gene and the CaMV35S promoter of rice transformed with Bt gene could be effectively amplified. Specific experimental results show that this method has high specificity and stable results. No GM samples were detected in commercially available samples. Conclusion Compared with conventional PCR, the multiplex PCR method for the determination of genetically modified rice established in this study is more rapid, simple, and more specific. It can be used for rapid and qualitative detection of transgenic rice.