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

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.010

Abstract

Abstract: 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 125 μmol/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.

Key words: multiplex PCR, flue-cured tobacco, GMO ingredient

Yu Jing, Guo Yushuang, Lin Shifeng, Yu Shizhou, Zhao Jiehong. A Multiplex PCR for Rapid Detection of Genetically Modified Ingredient in Flue-cured Tobacco[J]. Biotechnology Bulletin, 2015, 31(7): 64-68.

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