Novel Reference Gene, High-mobility-group protein I/Y, Used in Qualitative and Real-Time Quantitative Polymerase Chain Reaction Detection of Transgenic Rapeseed Cultivars

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Abstract:

With the development of transgenic crops, regulations to label the genetically modified organisms (GMOs) and their derived products have been issued in many countries. Polymerase chain reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods are generally needed to amplify the transgene and compare the amplified results with that of a corresponding reference gene to get the reliable results. Specific primers were developed for the rapeseed (Brassica napus), High-mobility-group protein I/Y (HMG-I/Y) single-copy gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 15 different rapeseed varieties, and identical amplified products were obtained with all of them. No amplification was observed when templates were the DNA samples from the other species of Brassica genus or other species, such as broccoli, stem mustard, cauliflower, Chinese cabbage, cabbage, sprouts, Arabidopsis thaliana, carrot, tobacco, soybean, mung bean, tomato, pepper, eggplant, plum, wheat, maize, barley, rice, lupine, and sunflower. This system was specific for rapeseed. Limits of detection and quantitation in qualitative and quantitative PCR systems were about 13 pg DNA (about 10 haploid genomes) and about 1.3 pg DNA (about 1 haploid genome), respectively. To further test the feasibility of this HMG-I/Y gene as an endogenous reference gene, samples containing transgenic rapeseed GT73 with the inserted glyphosate oxidoreductase (GOX) gene were quantitated. These demonstrated that the endogenous PCR detection systems were applicable to the qualitative and quantitative detection of transgenic rapeseed.

Document Type: Research Article

Affiliations: 1: Nanjing University, Department of Biological Science and Technology, 22 Hankou Rd, Nanjing 210093; Shanghai Jiao Tong University, School of Life Science and Biotechnology, 800 Dongchuan Rd, Shanghai 200240; Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotech Research Center, 2901 Beidi Rd, Shanghai 201106, People's Republic of China. 2: Nanjing University, Department of Biological Science and Technology, 22 Hankou Rd, Nanjing 210093, People's Republic of China. 3: Shanghai Jiao Tong University, School of Life Science and Biotechnology, 800 Dongchuan Rd, Shanghai 200240; Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotech Research Center, 2901 Beidi Rd, Shanghai 201106, People's Republic of China. 4: Shanghai Jiao Tong University, School of Life Science and Biotechnology, 800 Dongchuan Rd, Shanghai 200240; Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotech Research Center, 2901 Beidi Rd, Shanghai 201106, People's Republic of China. 5: Shanghai Jiao Tong University, School of Life Science and Biotechnology, 800 Dongchuan Rd, Shanghai 200240; Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotech Research Center, 2901 Beidi Rd, Shanghai 201106; Shanghai Teachers University, Life and Environment Science College, 10 Guilin Rd, Shanghai 200234, People's Republic of China.

Publication date: March 1, 2005

More about this publication?
  • The Journal of AOAC INTERNATIONAL publishes refereed papers and reviews in the fields of chemical, biological and toxicological analytical chemistry for the purpose of showcasing the most precise, accurate and sensitive methods for analysis of foods, food additives, supplements and contaminants, cosmetics, drugs, toxins, hazardous substances, pesticides, feeds, fertilizers and the environment available at that point in time. The scope of the Journal includes unpublished original research describing new analytical methods, techniques and applications; improved approaches to sampling, both in the field and the laboratory; better methods of preparing samples for analysis; collaborative studies substantiating the performance of a given method; statistical techniques for evaluating data. The Journal will also publish other articles of general interest to its audience, e.g., technical communications; cautionary notes; comments on techniques, apparatus, and reagents.
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