关键词: 转基因作物, 抗虫性, cry1A基因, 简并PCR, 筛选检测方法

Abstract: 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.

Key words: genetically modified crop, insect-resistance, cry1A gene, PCR with degenerate primers, screening and detection methods