title: Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods.
abstract: The Cauliflower mosaic virus (CaMV) 35S promoter (P35S) is a commonly used target for detection of genetically modified organisms (GMOs). There are currently 24 reported detection methods, targeting different regions of the P35S promoter. Initial assessment revealed that due to the absence of primer binding sites in the P35S sequence, 19 of the 24 reported methods failed to detect P35S in MON88913 cotton, and the other two methods could only be applied to certain GMOs. The rest three reported methods were not suitable for measurement of P35S in some testing events, because SNPs in binding sites of the primer/probe would result in abnormal amplification plots and poor linear regression parameters. In this study, we discovered a conserved region in the P35S sequence through sequencing of P35S promoters from multiple transgenic events, and developed new qualitative and quantitative detection systems targeting this conserved region. The qualitative PCR could detect the P35S promoter in 23 unique GMO events with high specificity and sensitivity. The quantitative method was suitable for measurement of P35S promoter, exhibiting good agreement between the amount of template and Ct values for each testing event. This study provides a general P35S screening method, with greater coverage than existing methods.
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Yuhua
authors.initials: Y
authors.lastname: Wu
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] College of Life Sciences, Hubei University, No. 368 Friendship Avenue, Wuhan 430062, People's Republic of China.
authors.forename: Yulei
authors.initials: Y
authors.lastname: Wang
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: School of Life Science, South-Central University for Nationalities, Min-Yuan Road 708, Wuhan 430074, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Yunjing
authors.initials: Y
authors.lastname: Li
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Li
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhu
authors.affiliation: 1] Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China [2] Supervision and Test Center (Wuhan) for Environmental Safety of Genetically Modified Plants, Ministry of Agriculture, No. 2 Xudong 2nd Road, Wuhan 430062, People's Republic of China.
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
mesh_terms: D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics*; D018556:Crops, Agricultural / Q000821:virology; D016208:Databases, Factual; D008969:Molecular Sequence Data; D009154:Mutation; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment