多重PCR检测耐除草剂转基因作物

Multiplex PCR Detection of Herbicide-tolerant Genes in Genetically Modified Crops

为建立耐除草剂转基因作物的高通量检测方法,本试验以目前生产上广泛应用的5种除草剂抗性基因dmo、pat、CP4EPSPS、bar和aad1为靶标进行多重PCR(MPCR)研究。通过引物适用性测试、反应体系中的不同引物浓度和反应程序中的退火温度测试、灵敏度和特异性验证等,建立了能同时检测5种除草剂抗性基因的MPCR检测方法。结果表明,当dmo、pat、CP4EPSPS、bar、aad1基因的检测引物终浓度分别为0.2、0.2、0.3、0.4、0.2μmol·L^(-1),退火温度为63℃时5种靶标扩增效果较好,且特异性条带清晰且均一。此外,MPCR检测方法具有较好的特异性,对每种靶标的检测灵敏度均可达到0.1%。适用性测试结果显示,MPCR检测方法可对含有5种除草剂抗性基因的多种转基因作物的单个品系或多个品系混合物进行筛选检测。无假阳性和假阴性结果表明,MPCR检测方法对实际样品具有很好的适用性。本试验结果为筛选高效耐除草剂转基因作物检测技术提供了一定的理论依据。

In order to establish a high-throughput detection method for herbicide-tolerant transgenic crops, five herbicide-tolerant genes, dmo, pat, CP4EPSPS, bar and aad1,which are widely used in production, were selected as targets in this study. By verifying the applicability of designed primers, determining the concentration of primers, annealing temperature, and the sensitivity and specificity of the detection system, the MPCR assay for detecting these five herbicide-tolerant genes simultaneously was developed. The results showed that when the final concentration of primers for dmo, pat, CP4EPSPS, bar and aad1 genes were 0.2, 0.2, 0.3, 0.4, 0.2 μmol·L^-1, respectively, and the annealing temperature was 63℃, these five target genes could be well amplified and the specific bands were clear and uniform. In addition, the pentaplex PCR method had good specificity, and the detection sensitivity for each target gene could reach 0.1%. The applicability testing showed that this MPCR method could be used to screen the target genes from single event or mixture of several events, and there was no false positive and false negative amplification, which indicated that the system had good applicability in practical application. The study provided a high-efficient screening mean for the detection of herbicide tolerant genetically modified crops.