柑橘转基因成分多重PCR检测体系的建立

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 2.5μl,25 mmol/L MgCl22.0μl;dNTP Mixture(2.5 mmol/L each)2.0μl,10μmol/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、0.5μl,模板DNA 0.1μg,Taq DNA聚合酶1.25U,加ddH2O至25μl。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。

Establishment of a Multiplex PCR System for Detecting Transgenic Ingredients from Citrus

Objective] This study aimed to establish a multiplex PCR system for detecting transgenic ingredients from Citrus.Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citrus,the primers specific to CaMV35S promoter,NOS promoter,NOS terminator and actin gene were designed,to establish a multiple PCR system which can detect four types of sequences.In addition,orthogonal tests were performed to determine the optimal concentrations of all the components in PCR reaction system,as well as the optimal PCR cycle parameters.Result] The optimal PCR reaction system should contain 2.5 μl of 10× PCR buffer,2.0 μl of MgCl2(25 mmol/L),2.0 μl of dNTP mixture(2.5 mmol/L of each dNTP),1.0 μl of actin gene primers(10 μmol/L),1.0 μl of 35S promoter primers(10 μmol/L),1.5 μl of NOS promoter primers(10 μmol/L) and 0.5 μl of NOS terminator primers(10 μmol/L),0.1 μg of template DNA,1.25 U of Taq DNA polymerase;ddH2O was added to the total reaction system of 25 μl.The PCR reaction program consisted of pre-denaturing at 94 ° C for 5 min;31 cycles of denaturing at 94 ℃ for 30 s,annealing at 64.1 ℃ for 45 s and extension at 72 ℃ for 50 s;final extension at 72 ℃ for 10 min.The reaction system optimized with the orthogonal tests could detect as less as 0.1% transgenic component in the tested samples.Conclusion] The MPCR detection system established in this study can meet the requirements in theory for detecting the genetically modified ingredients in Citrus or the deep-processed products.