CAJ | 학술논문

[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pBl 121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：10X buffer2．5μl，25mmol／LMgCl2 2．0μl；dNTP Mixture（2．5mmol／Leach）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DNA0．1μg，砌DNA聚合酶1．25u，加ddH2O至25μl。PCR反应程序为：94℃预变性5min；94℃30s，64．1℃45S，72℃50s，31个循环；72℃10min。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。
[목적]건립감귤전기인성분적다중PCR검측체계。[방법]근거GenBank중pBl 121질립서렬화감귤（Citrus．）Actin기인서렬，분별설계CaMV35S계동자、NOS계동자、NOS종지자특이인물화Actin기인적특이인물，건립능동시검측출4충서렬적다중PCR검측체계，동시통과정교시험학정해체계적최가인물농도화비례급PCR반응체계중각인소적농도급반응정서，병대해방법적령민도진행험증。[결과]시험득도적최가MPCR반응체계위：10X buffer2．5μl，25mmol／LMgCl2 2．0μl；dNTP Mixture（2．5mmol／Leach）2．0μl，10μmol／L적Actin기인、35S계동자、NOS계동자、NOS종지자인물분별가입1．0、1．0、1．5、0．5μl，모판DNA0．1μg，체DNA취합매1．25u，가ddH2O지25μl。PCR반응정서위：94℃예변성5min；94℃30s，64．1℃45S，72℃50s，31개순배；72℃10min。시험중，경정교우화후적4중PCR반응령민도체0．1％。【결론】해연구건립적MPCR검측체계，이론상이능만족감귤혹기심가공산품적전기인성분검측。
[Objective] This study aimed to establish a multiplex PCR system for de- tecting 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 could 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 2.5μl of 10xPCR buffer, 2.0μl of MgCI2 （25 mmol/L）, 2.0 μl of dNTP mixture （2.5 mmol/L of each dNTP）, 1.0 μl of actin gene primers （10μmol/L）, 1.0μl 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 （10μmol/L）, 0.1 μg of template DNA, 1.25 U of Taq DNA polymerase; ddH20 was added to the total reaction system of 25μl. The PCR reaction program consisted of pre-denaturing at 94℃ 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.