中国农业科学2012，Vol.45Issue(5)：1010-1016,7.DOI:10.3864/j.issn.0578-1752.2012.05.023
基于多重串联式PCR的基因碟片技术检测转基因作物
Multiplex Tandem PCR Assays for the Detection of Genetically Modified Organisms
摘要
Abstract
[Objective] The objective of this study is to develop a multiplex tandem polymerase chain reaction (MT-PCR) gene disk method for the detection of genetically modified organism (GMO). [ Method] The MT-PCR method was employed to detect NOS terminator, FMV35S promoter, alien genes including NPT1I, CrylAb, CrylAb/Ac, CP4-EPSPS and PAT, which was normally used in transformation, and endogenous genes such as IVR from maize, sadl from cotton, and PEP from rapeseed meal. Inner and outer primers were specially designed for each gene. Following the first PCR reaction, the multiplexed amplicons were simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product was then diluted and analyzed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR used a template enriched in the amplicons of interest, the conditions could be optimized to significantly reduce 'primer dimer' formation allowing SYBR Green chemistry to be used for quantification. The final results were achieved by cycling curves and melting curve. [ Result] The MT-PCR method was rapid (<2 h), high-throughput (10 genes of each sample), sensitive (>0.000292 ng), and specific in detecting GMO, such as cotton seeds, maize, rice and rapeseed meal. The method is making very fine distinctions about the three kinds of transgenic species can be made by this method and it fits for a large amount of detection. [ Conclusion ] The MT-PCR method is specific, stable and reliable, and will be an effective tool for the detection of GMO.关键词
转基因/多重串联式PCR/基因碟片技术Key words
genetically modified organism (GMO)/ multiplex tandem polymerase chain reaction (MT-PCR)/ gene disk引用本文复制引用
魏霜,陈贞,马骏,白卫滨,吴希阳..基于多重串联式PCR的基因碟片技术检测转基因作物[J].中国农业科学,2012,45(5):1010-1016,7.基金项目
农业部转基因重大专项“转基因生物检测和监测新技术”(2008ZX08012-005) （2008ZX08012-005）
