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[目的]建立柑橘转基因成分的多重 PCR 检测体系。[方法]根据 GenBank 中 pBI121 质粒序列和柑橘(Citrus.)Actin 基因序列,分别设计CaMV35S 启动子、NOS 启动子、NOS 终止子特异引物和 Actin 基因的特异引物,建立能同时检测出 4 种序列的多重 PCR 检测体系,同时通过正交试验确定该体系的最佳引物浓度和比例及 PCR 反应体系中各因素的浓度及反应程序,并对该方法的灵敏度进行验证。[结果]试验得到的最佳MPCR 反应体系为:10×buffer 2.5 μl,25 mmol/L MgCl22.0 μl;dNTP Mixture (2.5 mmol/L each)2.0 μl,10 μmol/L 的 Actin 基因、35S 启动子、NOS 启动子、NOS 终止子引物分别加入 1.0、1.0、1.5、0.5 μl,模板 DNA 0.1 μg,Taq DNA 聚合酶 1.25 U,加 ddH2O 至 25 μl。PCR 反应程序为:94 ℃预变性5 min;94 ℃ 30 s,64.1 ℃ 45 s,72 ℃ 50 s,31个循环;72 ℃ 10 min。试验中,经正交优化后的4重PCR反应灵敏度达0.1%。[结论]该研究建立的MPCR检测体系,理论上已能满足柑橘或其深加工产品的转基因成分检测。
[Objective] The research aimed to establish a multiplex PCR detection system of transgenic components of citrus. [Method] CaMV35S promoter, NOS promoter, NOS terminator specific primers and Actin gene specific primers were designed according to the sequence of pBI121 in GenBank and the actin gene sequence of citrus (Citrus) respectively. Four primers Multiplex PCR detection system was established. At the same time, the optimal primer concentration and proportion of the system and the concentration of each factor in the PCR reaction system were determined by orthogonal test. The sensitivity of the method was also verified. [Result] The optimum MPCR reaction system was as follows: 10 μl buffer 2.5 μl, 25 mmol / L MgCl 22.0 μl, 2.0 μl dNTP Mixture (2.5 mmol / L each), Actin gene at 10 μmol / L, NOS promoter, NOS terminator primers were added 1.0, 1.0, 1.5, 0.5 μl, template DNA 0.1 μg, Taq DNA polymerase 1.25 U, plus ddH2O to 25 μl. The PCR reaction procedure was as follows: pre-denaturation at 94 ° C for 5 min; 31 cycles of 94 ° C for 30 s, 64.1 ° C for 45 s, 72 ° C for 50 s; and 72 ° C for 10 min. In the experiment, the 4-fold PCR reaction after orthogonal optimization has a sensitivity of 0.1%. [Conclusion] The MPCR detection system established in this study could theoretically meet the detection of transgenic components of citrus or its processed products.