Event-specific qualitative and quantitative PCR detection of MON863 maize based upon the 3′-transgene integration sequence |
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| Institution: | 1. Key Laboratory of Agricultural Genetics and Breeding, Agro-biotech Research Center, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, People''s Republic of China;2. School of life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People''s Republic of China;3. Department of Biological Science and Technology, Nanjing University, 22 Hankou Road, Nanjing 210093, People''s Republic of China;4. Institute of Plant Protection, Chinese Academy of Agricultural Sciences, 12 Southern Zhongguancun Road, Beijing 100081, People''s Republic of China;1. Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 20040, China;2. Deparment of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK;3. School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, SA 5064, Australia;1. The Affiliated Hospital of Qingdao University, Qingdao 266003, China;2. Department of Pharmacology, Medical College, Qingdao University, Qingdao 266071, China;3. School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW 2650, Australia;1. British Heart Foundation Cardiovascular Sciences Unit, National Heart and Lung Institute, Imperial College London, UK;2. Department of Cardiovascular Sciences, University of Sheffield, Beech Hill Road, Sheffield S10 2RX, Sheffield, UK |
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| Abstract: | An event-specific detection method was developed based on the flanking sequence of an exogenous integrant in the transgenic maize MON863 which contains cry3Bb1 gene expressing a Bacillus thuringiensis Cry3Bb1 protein that is selectively toxic to a maize root worm pathogen. The 3′-integration junction between host plant DNA and integrated DNA of transgenic MON863 maize was isolated using thermal asymmetric interlaced (TAIL)-PCR. The event-specific primers and TaqMan probe were designed based upon the isolated 3′-integration junction sequence, and qualitative and quantitative PCR systems were established employing these designed primers and probe. In this system, the limit of detection of the qualitative PCR assay was estimated to be 40 initial haploid copies. The limit of quantitation of the quantitative PCR assay in authentic MON863 maize seeds was estimated to be approximately 80 haploid copies. GM MON863 contents were also quantified relative to endogenous maize starch synthase IIb (zSSIIb) gene DNA, and the results were expressed as the percentage of genetically modified MON863 maize DNA relative to the total content of maize DNA. All the results indicated that the established MON863 event-specific qualitative and quantitative PCR detection system based on the 3′-integration junction was reliable, sensitive and accurate. |
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