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

作     者：李政利 彭爱红 邹修平 何永睿 姚利晓 陈善春 

作者机构：西南大学园艺园林学院重庆400716 中国农业科学院柑桔研究所国家柑桔工程技术研究中心国家柑桔品种改良中心重庆400712 

基　　金：Supported by the Special Fund for Key Laboratories of Chongqing (CSTC) National Technology Research and Development Program of Ministry of Science and Technology for Countryside Field (863 Program,2011AA100205) Special Fund for Agro-scientific Research in the Public Interest of Ministry of Agriculture of China(201003067) Key Science and Technology Research Program of Ministry of Education of China (109131) 

出 版 物：《Agricultural Science & Technology》 (农业科学与技术（英文版）)

年 卷 期：2012年第13卷第5期

页      码：952-957页

摘      要：[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.

主 题 词：Multiplex PCR Orthogonal test Detection Genetically modified ingredients 

学科分类：09[农学] 0902[农学-自然保护与环境生态类] 090201[090201] 

D　O　I：10.3969/j.issn.1009-4229-B.2012.05.008

馆 藏 号：203334421...