Multiplex PCR detection of herbicide-tolerant genes in genetically modified crops

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. (authors)