荧光定量PCR法定量检测混合烟叶中的红花大金元  

Quantitative detection of cv.Honghuadajinyuan in mixed tobacco leaves by fluorescent quantitative PCR

作　　者：李萌 候宁宁 曲鹏 王旭东 宋嘉宝 孙九喆 马林 LI Meng;HOU Ningning;QU Peng;WANG Xudong;SONG Jiabao;SUN Jiuzhe;MA Lin(College of Food Science and Bioengineering,Zhengzhou University of Light Industry,Zhengzhou 450001,China;Technology Center,China Tobacco Henan Industrial Co.,Ltd.,Zhengzhou 450000,China)

机构地区：[1]郑州轻工业大学食品与生物工程学院,郑州450001 [2]河南中烟工业有限责任公司技术中心,郑州市450000

出　　处：《烟草科技》2022年第1期25-32,共8页Tobacco Science & Technology

基　　金：河南中烟工业有限责任公司科技计划项目“黄金叶品牌烟叶品种DNA指纹图谱库建立的应用研究”(ZW2015012)。

摘　　要：为检测混合烟叶中红花大金元烟叶,设计了红花大金元品种特异性引物与TaqMan MGB探针,检测了引物和探针的特异性和灵敏性,确定了荧光定量PCR反应条件,并在此基础上建立标准曲线对混合烟叶中红花大金元单一品种进行定量检测。结果表明,引物与探针组合H1-R/F/T的特异性较好,建立了标准曲线方程,决定系数R2为0.997。当DNA(55 ng·μL-1)模板含量为2μL时,红花大金元的检测限可达0.11 ng。利用该方法对混合烟叶样品中红花大金元含量进行检测,误差范围在2.5%~3.5%之间,表明该方法可应用于特定混合样品中红花大金元烟叶的定量检测。In order to quantitatively detect Honghuadajinyuan in mixed tobacco leaves,cultivar-specific primers and TaqMan MGB probes were designed.The specificity and sensitivity of the primers and the probes were detected,and the reaction conditions of the fluorescent quantitative PCR were determined.On this basis,a regression equation was established to quantitatively detect the single cultivar of Honghuadajinyuan in mixed tobacco leaves.The results showed that the combination of the primers and the probes H1-R/F/T had satisfactory specificity.The regression equation was established and the determination coefficient R2 was 0.997.When the template DNA(55 ng·μL-1)was 2μL,the detection limit of Honghuadajinyuan was 0.11 ng.The error range of the content of Honghuadajinyuan in mixed tobacco leaves detected by this method was between 2.5%and 3.5%,suggesting that this method could be applied to the quantitative detection of Honghuadajinyuan in specific mixed tobacco leaves.

关 键 词：红花大金元 烟叶 荧光定量PCR TaqMan MGB探针 烟叶品种识别 

分 类 号：S572.02[农业科学—烟草工业]