水稻种子样品前处理对转基因成分荧光PCR检测的影响  

Effect of pretreatment of rice seed samples on real-time fluorescence PCR detection of genetically modified ingredients

作　　者：王志纯 张冰[2,3] 邵碧英[2] 江树勋[2] 缪婷玉[2] 彭娟[2] 陈文炳[2] 

机构地区：[1]福建省种子管理总站,福建福州350003 [2]福建出入境检验检疫局检验检疫技术中心,福建福州350001 [3]厦门塔斯曼生物工程公司,福建厦门361023

出　　处：《福建农林大学学报（自然科学版）》2015年第1期8-13,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建出入境检验检疫局科技计划项目(FK2010-28;FK2013-02)

摘　　要：以转基因水稻科丰6号不同含量的水稻种子为样品,对影响检测结果的主要前处理措施,包括样品研磨颗粒细度与DNA提取过程中样品CTAB裂解缓冲液温育时间等,进行分析筛选.结果表明,样品颗粒细度与CTAB缓冲液温育时间的不同处理组合对提取的DNA含量影响极显著,其中,样品颗粒细度>100目与CTAB缓冲液温育时间>8 h(过夜)处理的样品DNA提取效果与荧光PCR检测结果均最佳.采用最佳前处理组合,样品的主要转基因成分(Ca MV 35S、NOS、Cry1Ab)检出限从通常的转基因含量质量分数0.01%降到0.001%,使得水稻种子转基因成分荧光PCR检测灵敏度提高10倍,大幅度提高了检测结果的准确性.To improve the sensitivity and detection rate of fluorescent real-time PCR（ RT-PCR） detecting transgenic components in rice seeds,pretreatment measures including sample particle fineness,hexadecyltrimethyl ammonium bromide（ CTAB） lysis buffer incubating time to extract DNA were screened using rice seed samples containing transgenic rice Kefeng 6. The results showed that the content of DNA has significant differences among different treatment combinations between sample particle fineness and CTAB lysis buffer incubating time,in which,the content of DNA extracted by the combination of sample particle fineness 〉 100 mesh and CTAB lysis buffer incubating time 〉 8 hours（ overnight） was the highest; the fluorescent RT-PCR detection results obtained with sample treatment combination mentioned,was ideal. The detection limit of major transgenic components in a sample was reduced from transgenic content（ Ca MV 35 S,NOS,Cry1Ab） 0. 01%（ m / m） recognized to 0. 001%（ m / m）,i. e. the sensitivity of detection method was improved by 10 times,using the optimal pretreatment combination. The accuracy of the fluorescent RT-PCR detection results of genetically modified ingredients in rice seed was improved effectively.

关 键 词：水稻种子 转基因成分 实时荧光PCR 检出限 

分 类 号：S339.3[农业科学—作物遗传育种]