基于多重串联式PCR的基因碟片技术检测转基因作物

doi:10.3864/j.issn.0578-1752.2012.05.023

摘要/Abstract

摘要： 【目的】建立基于多重串联式PCR（multiplexed tandem PCR，MT-PCR）的基因碟片技术，并使之应用于转基因作物的大量、快速和稳定地检测。【方法】以转基因作物研究中常用的调控序列NOS终止子、FMV35S启动子及外源基因NPTⅡ、Cry1Ab、Cry1Ab/Ac、CP4-EPSPS、PAT和玉米内源基因IVR、棉花内源基因sad1、菜籽粕内源基因PEP为检测对象，针对每个基因设计内外2对引物，先进行一次循环数较少（10—20 cycles）的高通量多重PCR，以便在均匀地扩增各基因和调控序列的同时避免引物之间的竞争，然后利用巢式荧光定量PCR检测各个基因和调控序列，最后根据扩增曲线和熔融曲线分析结果。【结果】该方法能够快速（＜2 h）、高通量、准确地（＞0.000292 ng）检测出棉花、玉米、大米、菜籽粕中的多种转基因成分，可以分辨出3种转基因物种，适合大批量检测。【结论】该方法适合转基因作物的高通量、定量检测，具有较好的应用前景。

关键词: 转基因, 多重串联式PCR, 基因碟片技术

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 NPTⅡ, Cry1Ab, Cry1Ab/Ac, CP4-EPSPS and PAT, which was normally used in transformation, and endogenous genes such as IVR from maize, sad1 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.

Key words: genetically modified organism (GMO), multiplex tandem polymerase chain reaction (MT-PCR), gene disk

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.

WEI Shuang, CHEN Zhen, MA Jun, BAI Wei-Bin, WU Xi-Yang. Multiplex Tandem PCR Assays for the Detection of Genetically Modified Organisms[J]. Scientia Agricultura Sinica, 2012, 45(5): 1010-1016.

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https://www.chinaagrisci.com/CN/Y2012/V45/I5/1010

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