转基因番茄Zeneca B,Da,F实时荧光定量PCR检测体系的建立  

Construction of quantitative real-time PCR detection system of transgenic tomato line Zeneca B,Da,F

作　　者：麦荣嘉[1] 陈敏芳[1] 莫倩珍[1] 胡良勇[2] 黎事伦[1] 唐敏然[2] 顾晓敏[1] 蔡永洪[2] 周伦彬[2] 周晓红[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广东广州510515 [2]广州市计量检测技术研究院,广东广州510030

出　　处：《南方医科大学学报》2011年第4期587-593,共7页Journal of Southern Medical University

基　　金：国家科技支撑计划(2008BAK41B01);国家自然科学基金(30872204;81071378);广东省自然科学基金(5004747)~~

摘　　要：目的构建转基因番茄Zeneca B,Da,F品系的质粒标准分子及其实时荧光定量PCR(qPCR)检测体系。方法基于反向遗传学原理构建分别含有番茄内标准基因apx检测序列(ERG-apx),转基因番茄B,Da,F品系pg基因的基因特异性序列(GS-pg)及其构建特异性序列(CS-16S、CS-16A)的番茄内参质粒标准分子pEasy-T3-APX,转基因番茄B,Da,F品系的构建特异性质粒标准分子pEasy-T3-16A、pEasy-T3-16S;以Beacon Designer 7.5设计用于定性PCR和qPCR检测的引物对和Taqman探针;基于质粒标准分子建立了定性PCR和qPCR检测体系,对方法的特异性、敏感性、重复性、检测下限等指标进行评估;使用PicoGreen试剂盒对质粒标准分子进行定量,以质粒pEasy-T3-APX为背景DNA定量混有pEasy-T3-16A或pEasy-T3-16S制备成含量为1%和0.1%(w/w)的两组核酸标准物质,进行所构建qPCR检测体系的定量性能评价。结果锚定qPCR检测靶序列:ERG-apx 108 bp,GS-pg 108 bp,CS-16S 109 bp、CS-16A 102 bp;酶切鉴定所构建的质粒标准分子均可得到预期大小的插入子片段;所建立的实时荧光定量PCR(qPCR)检测体系,重复性的相对标准差(RSD)0.2%～1.5%,检测下限25 copies,标准曲线方程线性关系R2值在0.994～0.998,效率在93.3%～102.4%。经换算系数Cf换算,对PicoGreen定量的样品进行验证,其实测值与真值的偏差是-9.7%～17.3%。结论成功构建了转基因番茄Zene-ca B,Da,F品系的质粒标准分子及其qPCR检测体系,为转基因番茄Zeneca B,Da,F品系转基因成分的监测建立了可行的可供使用的定性与定量检测体系。To construct the plasmid reference molecules for detection of transgenic tomato line Zeneca B,Da,F using quantitative real-time PCR（qPCR）.Methods Three plasmid reference molecules pEasy-T3-APX,pEasy-T3-16A and pEasy-T3-16S were cloned based on reverse genetics,which contain the target fragments of tomato endogenous reference gene apx（ERG-apx）,gene-specific sequence of pg（GS-pg） and construct-specific sequence of vectors pJR16S/pJR16A（CS-16S/CS-16A） of Zeneca B,Da,F,respectively.Primers and Taqman probes were designed by Beacon Designer 7.5.The specificity,sensitivity,reproducibility and the limit of detection（LOD） of the qualitative and quantitative PCR system based on the plasmid reference molecules were evaluated.PicoGreen was used to measure the DNA concentration of the plasmid reference molecules.Two sets of samples containing 1% or 0.1%（w/w） pEasy-T3-16A or pEasy-T3-16S mixed with pEasy-T3-APX as background DNA were prepared for evaluating the efficacy of the qPCR system.Results The target fragments for qPCR detection were anchored,ERG-apx 108 bp,GS-pg 108 bp,CS-16S 109 bp and CS-16A 102 bp.The three plasmid reference molecules were confirmed at the expected sizes by restriction enzyme digestion.The qPCR results showed that the RSD of reproducibility were 0.2% to1.5%,LOD was 25 copies,R2 values for these standard curves were 0.994 ～0.998 and amplification efficiencies were 93.3%～102.4%.The bias between the test and true values of two sets of mixed samples ranged from-9.3% to 14.7% after adjusting by conversion factors（Cf）.Conclusion The plasmid reference molecules and qPCR system for qualitative and quantitative detection of transgenic tomato line Zeneca B,Da,F have been established successfully.

关 键 词：质粒标准分子 转基因番茄 核酸检测技术 荧光定量PCR 

分 类 号：Q943.2[生物学—植物学] S641.2[农业科学—蔬菜学]