关键词: 实时荧光PCR, 转基因大豆, Taqman探针, SYBR染料, 定量检测

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.

Key words: Real-time PCR, Genetically modified soybean, Taqman probe, SYBR dye, Quantitative detection