Construction of a General Standard Molecule for the Qualitative Detection in Different Transgenic Foodcrops

Now every laboratory is facing a common problem that is seriously lacking in the standard reference molecule in the GMC detecting research center. Constructing the recombinant plasmid is the urgent demand. In order to overcome this problem, this study respectively cloned CrylA（B）, BAR, CP4-EPSPS, PAT and RBCL genes into PMD 18-T vector. This constructed plasmid could be used as the standard reference molecule in the qualitative detecting of the exogenous CP4-EPSPS gene, CrylA（B） gene, BAR gene and PAT gene. Identification by double restriction endonuclease digestion and identification by PCR proved that the test result was positive. All of these indicated that the established reference molecules in this study were suitable for the qualitative analysis standard of this GMC detection.

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x＋35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x＋35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

Comparison of Two Real-time PCR Technigues for Quantification of GMO Contents in Highly Processed Products of Soybean

The RR soybean was quantitatively detected by ABI Prism 7300 sequence detector with PCR primers and fluorescence probes were designed according to the sequences of endogenous Lectin gene and exogenous CP4-EPSPS gene, and the PCR systems were based on SYBR Green I and TaqMan. The standard curve of ACt between CP4-EPSPS gene and Lectin gene of the RR soybean in standard materials was generated and a linear regression equation was obtained. Quantification methods were optimized through two different real-time PCR chemistries, i.e. SYBR Green I and TaqMan, and the RR soybean contents were quantified in five standard samples and seven highly processed products by the two assays. Both methods are proved to be specific, highly sensitive and reliable for both identification and quantification of soybean DNA. The results indicate that the two optimized PCR system can be used for the practical quantitative detection of RR soybean in highly processed products.