| Genetically modified crop is developing rapidly, since1996it has been planted for commercialization. Genetically modified organisms (GMO) are referred as living organisms of crop origin in which the artificial introduction of foreign genes are able to confer novel characteristics not found in the wild variety. Specificity, sensitivity and accuracy of GMO detecting techniques will be required strictly. By Chromosome walking PCR technique, the3’ flanking sequence of the exogenous gene in the pAHC25plasmid of wheat ’B73-6-1’was successfully cloned. The detection technique system of B73-6-1was established.In this study we contrysted the thermal asymmetric interlaced PCR (tail-pcr) and ligation mediated PCR two chromosome walking technoiques, the results showed that the connector-mediated chromosome walking was more suitable for this study to obtain the flanking sequence. An event specific transgenic detection method for ’B73-6-1’ was established by PCR with the specific primers based on the3’border. The amplification fragment include part sequence of both the exogenous DNA and wheat genome. The bioinformatics analysis revealed that the2538bp before the sequence from the vector backbone, the remainder was not found with the homologous sequence of wheat genome. Suggesting that the2.2kb sequence should be generated when the bar gene integrated into the wheat genome combined sequence and the unknown sequence of the wheat genome. Primers were designed upon the flanking sequence for establishing strain-specific qualitative PCR detection method of B73-6-1. This assay was applied to detect typical genetically modified crops, with high specificity for B73-6-1. We used real-time fluorescence quantitative PCR technique to detect the bar gene copy number in B73-6-1. Non-transfer genome of wheat DNA was used as an internal reference gene, and wx012as the endogenous reference gene, to calculate the copy number. Also, by real-time fluorescence quantitative PCR, the copy number of bar was detected and the copy number was11.The qualitative and quantitative strain-specific detecting method was constructed. This study also paid attention to specificity, reproducibility and sensitivity of these methods. The sensitivity was0.1%by the qualitative PCR method and lowest limit was0.01%by the quantitative PCR method.In this study, by the event specific qualitative PCR techniques, A4.7kb fragment in the flanking of3’pAHC25plasmid were amplified. Both qualitative and quantitative methods for the B73-6-1event-specific detection were established, the system was reproducible, showing a good potential for routine analytical use. It helps to provide the important guarantees for the inspection of import and export products. |
