| 实时荧光PCR技术定量检测转基因大豆方法的研究 |
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| 引用本文： | 吴影,宋丰顺,陆徐忠,赵伟,杨剑波,李莉.实时荧光PCR技术定量检测转基因大豆方法的研究[J].作物学报,2007,33(10):1733-1737. |
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| 作者姓名： | 吴影 宋丰顺 陆徐忠 赵伟 杨剑波 李莉 |
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| 作者单位： | 1 安徽省农科院水稻研究所，安徽合肥230031；2 安徽农业大学生命科学学院，安徽合肥230036 |
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| 摘 要： | 以转基因大豆Roundup Ready为材料，通过特异性引物和探针，对转基因大豆中外源基因cp4-epsps进行了定量检测。研究发现Taqman荧光探针法和SYBR荧光染料法均可作为转基因大豆定量检测的方法，但前者比后者具更高的检测灵敏度和精确度，其检测阈值可降到0.05%，变异系数为0.09%~0.53%。在此基础上，建立和优化了Taqman探针实时荧光定量PCR检测技术体系，有助于提高转基因作物及产品的生物安全性定量检测的准确度和可靠性。
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| 关 键 词： | 实时荧光PCR 转基因大豆 Taqman探针 SYBR染料 定量检测 |
| 收稿时间： | 2007-01-25 |
| 修稿时间： | 2007-01-25 |
Detecting Genetically Modified Soybean by Real-time Quantitative PCR Technique |
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| WU Ying,SONG Feng-Shun,LU Xu-Zhong,ZHAO Wei,YANG Jian-Bo,LI Li.Detecting Genetically Modified Soybean by Real-time Quantitative PCR Technique[J].Acta Agronomica Sinica,2007,33(10):1733-1737. |
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| Authors: | WU Ying SONG Feng-Shun LU Xu-Zhong ZHAO Wei YANG Jian-Bo LI Li |
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| Institution: | 1.Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230031, Anhui;2.Department of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui, China |
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| Abstract: | In this paper,fluorescence-labeled Taqman probes and SYBR dye were chosen to detect the amplified DNA fragments by PCR.Before special primers and probes were used to amplify the exogenous gene cp4-epsps,the endogenous gene Lectin was detected to avoid the fake negative result.Then,two quantitative systems were optimized.And the standard curve of Ct vs.the GM content in the reference materials was generated and a linear regression equation was obtained to quantify GM soybean.The result showed that fluorescence signal appeared at the 24th cycles in Taqman-labeled,while at the 18th cycles in SYBR-labeled.It suggested that a little primer dimmers or other unspecific amplifications had cumulated before objective product formed in SYBR assay.The correlation coefficient(R2=0.993) of SYBR assay was lower than that of Taqman(R2=0.999).Finally,the two quantitative systems were tested respectively by using known samples with three GM contents.The results indicated that the precision of two systems were high,and the recurrences of the results were fine.Comparing the two quantitative assays,it has higher delicacy and precision in Taqman assay.The inferior limit of detection was less than 0.05%,and the Coefficient Variance was up to 0.09%.A very sensitive quantitative PCR method for the detection of genetically modified(GM) soybean was developed and validated.We also discussed the difference between Taqman assay and SYBR assay on detecting GMOs,and recommend the Taqman assay to use in transgenic product detection,especially in GMO food detection. |
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| Keywords: | Real-time PCR Genetically modified soybean Taqman probe SYBR dye Quantitative detection |
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