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当前位置: 首页 > 文章 > 应用简并PCR方法检测转cry1A基因作物 食品科学 2018 (14) 317-322
Position: Home > Articles > Detection of Genetically Modified Crops with cry1A Gene by PCR with Degenerate Primers FOOD SCIENCE 2018 (14) 317-322

应用简并PCR方法检测转cry1A基因作物

作  者：
李葱葱;闫伟;夏蔚;董立明;龙丽坤;李飞武
单  位：
吉林省农业科学院农业质量标准与检测技术研究所
关键词：
转基因作物;抗虫性;cry1A基因;简并PCR;筛选检测方法
摘  要：
根据不同转基因作物中cry1A基因的保守区序列,建立简并聚合酶链式反应(polymerase chain reaction,PCR)对转cry1A基因作物检测方法。cry1A基因(包括cry1Ab、cry1Ac、cry1Ab/Ac、cry1A.105、mcry1Ac)是抗虫转基因作物中使用频次最高的目的基因,常用作转基因成分筛选检测的靶标。应用Vector NTI Advance 11.5软件将已报道的20余种cry1A基因PCR检测方法中的引物与11个cry1A基因序列进行比对,结果显示,这些方法的引物序列不能同时与所有cry1A基因序列完全配对,每对引物的错配碱基数均大于3个,且许多错配发生在引物的3’端,表明这些cry1A基因检测方法理论上仅适用于部分特定的靶标基因。在对MON810、Bt11、Bt176等11种转基因作物中cry1A基因核苷酸序列进行比对分析基础上,以其5’端的保守核苷酸序列为模板,筛选到1对简并引物,建立了cry1A基因的简并PCR方法。应用该方法能特异性地检测11种转基因作物中的cry1A基因,检测灵敏度可稳定达到0.1%。该方法为转基因作物中cry1A基因的高效筛选检测提供了一种新的技术手段。
译  名：
Detection of Genetically Modified Crops with cry1A Gene by PCR with Degenerate Primers
作  者：
LI Congcong;YAN Wei;XIA Wei;DONG Liming;LONG Likun;LI Feiwu;Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences;
单  位：
LI Congcong%YAN Wei%XIA Wei%DONG Liming%LONG Likun%LI Feiwu%Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences
关键词：
geneticaly modified crop;;insect-resistance;;cry1A gene;;PCR with degenerate primers;;screening and detection methods
摘  要：
A PCR with degenerate primers method was established for the detection of genetically modified crops with cry1A gene according to its conservative region sequence. The cry1A genes, cry1Ab, cry1Ac, cry1Ab/Ac, cry1A.105, and mcry1Ac, the most frequently used genes in insect-resistant transgenic crops, are commonly used as targets for screening genetically modified ingredients. In the present work, using Vector NTI Advance 11.5 software, we aligned the primers used for more than 20 conventional or real-time PCR methods for detecting the cry1A genes with the whole sequences of 11 collected cry1A genes. The results revealed that all of the primer pairs could not be completely matched with the cry1A gene sequences. There were more than three mismatched bases for each primer, and the mismatching occurred frequently at the 3'-end of the primer, which indicates that the reported cry1A gene detection methods are only suitable for some specific target genes. Based on the nucleotide sequence analysis of 11 cry1A genes from transgenic crops such as MON810, Bt11 and Bt176, a pair of degenerate primers were screened out to develop a PCR assay using the conserved nucleotide sequence at the 5'-end as template. The PCR assay developed showed high specificity and sensitivity with a relative limit of detection(LOD) of 0.1% and could offer an efficient method for screening cry1A genes in genetically modified crops.

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