多重PCR技术快速检测烤后烟叶转基因成分

A Multiplex PCR for Rapid Detection of Genetically Modified Ingredient in Flue-cured Tobacco

为提高烟叶转基因成分检测效率,建立了烤后烟叶中3种外源基因成分的多重PCR检测技术体系,通过优化该反应体系中的Mg2+、d NTP、r Taq酶、引物浓度及退火温度等参数,实验最适条件为将4对相等摩尔浓度引物预先按1∶1∶1∶1(V/V)混合,在Mg2+为1.5 mmol/L、d NTP为125μmol/L、r Taq酶1 U、混合引物1μmol/L,DNA 100 ng,退火温度65℃,循环数35个的反应条件下,在一次PCR反应中便可同时检测烟草内源基因NR,外源基因35S启动子、NOS终止子、NPT II筛选标记基因。优化后的反应体系能够有效地检测出转基因烤后烟叶百分比含量为0.9%(V/V)的转基因成分。

In order to improve the efficiency of detecting genetically modified ingredient （ GMI ） , we established a multiplex PCR detecting 3 exogenous gene ingredients in flue-cured tobacco. By optimizing the concentrations of Mg2＋, dNTP, rTaq, and primer as well as annealing temperature in the multiplex PCR system, the result showed that the optimal conditions for the experiment were ： 4 pairs of primers pre-mixed in ratio of 1 ： 1 ： 1 ： 1 （ V/V ） , Mg2＋ 1.5 mmol/L, dNTP 125μmol/L, rTaq polymerase 1 U, mixed primers 1 μmol/L, DNA 100 ng annealing temperature 65℃, and 35 reaction cycles ; under the above conditions, the system detected 1 tobacco endogenous gene NR, exogenous gene 35S promoter, NOS terminator and selectable marker NPT II gene by 1 PCR reaction. In this study, the optimized multiplex PCR could efficiently detect the GMI of 0.9% （ V/V ） in transgenic flue-cured tobacco.