cotton-specific gene, stearoyl-ACP desaturase, used as a reference for qualitative and real-time quantitative polymerase chain reaction detection of genetically modified organisms

• Published in: Journal of the science of food and agriculture Vol. 86; no. 7; pp. 1103 - 1109
• Main Authors: Xu, W.T, Huang, K.L, Wang, Y, Zhang, H.X, Luo, Y.B
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.05.2006; Wiley; John Wiley and Sons, Limited
• Subjects: Biological and medical sciences; Biotechnology; Cotton; Daucus; detection; detection limit; food contamination; Food industries; Fundamental and applied biological sciences. Psychology; Gene expression; General aspects; genetic markers; genetically modified organisms; Gossypium hirsutum; Helianthus; Hordeum vulgare; Lycopersicon esculentum; Malus; Methods of analysis, processing and quality control, regulation, standards; Musa; new methods; Oryza sativa; oxidoreductases; polymerase chain reaction; Prunus; Quantitative genetics; real-time quantitative polymerase chain reaction; sad1 gene; Solanum tuberosum; species differences; stearoyl-ACP desaturase; transgenes; transgenic plants; Zea mays; Reference; Use; cotton; Real time; Polymerase chain reaction; stearoyl-ACP desaturase; Analysis method; Gene; sad1 gene; Control method; genetically modified organisms; Molecular biology; Detection; Genetically modified organism
• ISSN: 0022-5142; 1097-0010
• DOI: 10.1002/jsfa.2464

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)-based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl-ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real-time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties.