title: Multiplex event-specific qualitative polymerase chain reaction for detecting three transgenic rice lines and application of a standard plasmid as a quantitative reference molecule.
abstract: The three most well-known genetically modified (GM) rice lines in China are TT51-1, KMD1, and KF6. The purposes of this study were to establish a multiplex event-specific qualitative polymerase chain reaction (meqPCR) system for simultaneous detection of the three transgenic rice events and to construct a plasmid as the reference molecule for quantitative analysis. Event-specific primers for each event were selected or designed by focusing on the transgene borders between the inserted DNA and the flanking rice DNA. The developed meqPCR was anticipated to detect distinct amplicons as 454, 398, 301, and 250bp from KF6, KMD1, TT51-1, and the rice endogenous reference gene, respectively. The robustness of the meqPCR was tested with different levels of the three transgenic rice genomic DNAs, and the sensitivity threshold of the meqPCR was at least 50ng of 0.1% rice DNA for each event when the three transgenic rice events present and with other GM materials together. The constructed plasmid was evaluated using mixed samples with known GM contents in real-time quantitative PCR. The results indicated that the constructed plasmid was acceptable and suitable for GM rice quantitative analysis.
authors.affiliation: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China; Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Xiaofu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Chen
authors.affiliation: Institute of Quality and Standard for Agricultural Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
authors.forename: Junfeng
authors.initials: J
authors.lastname: Xu
authors.affiliation: College of Chemistry and Life Science, Shenyang Normal University, Shenyang 110034, China.
authors.forename: Pengfei
authors.initials: P
authors.lastname: Wang
authors.affiliation: College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: wbshenh@njau.edu.cn.
authors.forename: Wenbiao
authors.initials: W
authors.lastname: Shen
mesh_terms: D001483:Base Sequence; D018744:DNA, Plant; D017343:Genes, Plant; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000235:genetics*; D010957:Plasmids
chemical_list: D018744:DNA, Plant
keywords: Genetically modified rice; KF6; KMD1; Multiplex PCR; Plasmid; TT51-1