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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。
Objective To construct plasmid standard molecules of transgenic tomato Zeneca B, Da, F and their real - time fluorescence quantitative PCR (qPCR) detection system. Methods Based on the principles of reverse genetics, the gene-specific sequences (GS-pg) and their sequence-specific sequences (pG) of apx detection sequence (ERG-apx) and transgenic tomato B, CS-16S and CS-16A), and pEasy-T3-16A and pEasy-T3-16S, respectively, were constructed using the standard plasmid pEasy-T3-APX and the transgenic tomato B, Da and F lines. 7.5 Designed primer pairs and Taqman probes for qualitative PCR and qPCR detection. Based on the plasmid standard molecules, established qualitative PCR and qPCR detection system to evaluate the specificity, sensitivity, repeatability and detection limit of the method. The picoGreen kit was used to quantify the plasmid standard molecules and pEasy-T3-APX was used as the background DNA to quantitatively mix with pEasy-T3-16A or pEasy-T3-16S to prepare two levels of 1% and 0.1% (w / w) Group of nucleic acid standard material, quantitative performance evaluation of the constructed qPCR detection system. Results The target sequence of qPCR was 108 bp, 108 bp in GS-pg, 109 bp in CS-16S and 102 bp in CS-16A. The restriction fragment length ; The established real-time quantitative PCR (qPCR) detection system, the relative standard deviation of repeatability 0.2% ~ 1.5%, the detection limit of 25 copies, the standard curve equation R2 linear relationship between 0.994 ~ 0.998, the efficiency of 93.3% ~ 102.4%. After the conversion factor Cf conversion, PicoGreen quantitative sample validation, in fact, the measured value and the true value of the deviation is -9.7% ~ 17.3%. Conclusion The plasmid standard molecules and their qPCR detection system of transgenic tomato Zeneca cassava B, Da and F lines were successfully constructed. The feasible and applicable qualitative and quantitative methods for the detection of transgenic components of Zeneca B, Da and F lines were established. Detection system.