应用多重PCR技术筛选检测转Bt基因作物

Screening Detection of Bt Genes in Genetically Modified Crops Using a Multiplex PCR Assay

摘要:

Bt基因在抗虫转基因作物中广泛应用，是转基因食品筛选检测中最主要的目的基因。本研究依据核苷酸序列分析结果，将在转基因作物中常见的8种Bt基因（cry1Ab、cry1Ac、cry1Ab/Ac、cry1A.105、cry1Ac-M、cry2Ab、cry3A和cry3Bb）划分为cry1A、cry2A、cry3A三个组，并根据每个组的一致性序列设计了可分别检测各组Bt基因的特异性检测引物，其中cry1A组采用了简并引物，经过特异性、灵敏度等测试，建立了针对cry1A组、cry2A组和cry3A组的单一PCR检测方法。此外，通过将这三对检测引物放入同一PCR体系中，还建立了可特异检测上述3组Bt基因的三重PCR方法。结果表明，本研究建立的单一PCR和三重PCR方法均可从各类样品中准确检测出预期Bt基因成分，检测灵敏度达到0.1%。本方法特异性强、灵敏度高，在转基因成分的筛选检测中有很好的应用前景。

Abstract:

Since Bt genes were widely used in insect-resistant transgenic crops, they became the most important target genes in screening genetically modified food. Based on nucleotide sequences analysis, eight common Bt genes (cry1Ab, cry1Ac, cry1Ab/Ac, cry1A.105, cry1Ac-M, cry2Ab, cry3A and cry3Bb) in transgenic crops were divided into cry1A, cry2A and cry3A groups. To detect three groups of Bt genes, degenerate primers for cry1A group and specific primers for cry2A and cry3A groups were designed according to the consensus sequences of each group, and three singlet PCR methods for each group respectively were established after testing specificity and sensitivity in this study. In addition, using the three pairs of PCR primers, a triplex PCR method also established to screen the three groups of Bt genes simultaneously. The results revealed that these methods could accurately detect the targeted Bt genes from the various transgenic samples, and the detection sensitivity was up to 0.1%. As the method established in this study showed highly specific and sensitive, it would be a good prospect in the screening of genetically modified organism.