四重PCR检测抗除草剂转基因油菜T45

Quadruple PCR detection of herbicide-resistant transgenic rapeseed T45

  • 摘要: 【目的】建立多重PCR检测抗除草剂转基因油菜T45的方法,为其转基因检测提供技术支持。【方法】选择油菜内源参照基因CruA、外源基因P-CaMV 35S、T-CaMV 35S和pat作为四重PCR检测基因,特异性引物序列参照国家相关标准或文献,通过对多重PCR退火温度和引物浓度的优化、灵敏度测试及已知样品验证,建立抗除草剂转基因油菜T45多重PCR检测体系。【结果】多重PCR的适宜退火温度为58℃,适宜引物终浓度(μmol/L)配比CruA∶P-CaMV 35S∶T-CaMV35S∶pat为0.1∶0.2∶0.2∶0.2,方法灵敏度为0.01 ng/μL,所有已知样品的扩增条带与其分子特征显示的外源基因元件完全一致。【结论】建立的抗除草剂转基因油菜T45品系四重PCR检测方法可实现在一个反应体系中同时检测抗除草剂转基因油菜T45内源参照基因和多个外源基因成分,为转基因油菜T45提供了一种有效的检测手段。

     

    Abstract: ObjectiveA multiple PCR detection met hod for herbicide-resistant transgenic rapeseed T45 was estab-lished, in order to provide a valuable reference method for detecting herbicide-resistant transgenic rapeseed T45.MethodThe endogenous gene CruA, three exogenous genes P-CaMV 35S, T-CaMV 35S and pat of rapeseed were se-lected as targets for quadruple PCR detection system, their specific primers were referenced to China national standards or reported literature. Then the reaction conditions, including annealing temperature and primer concentration, were opti-mized, the specificity and sensitivity were tested, and this system was validated by the known sample. Based on these re-searches, the quadruple PCR system for detecting herbicide-resistant transgenic rapeseed T45 were established. ResultThe results showed that, the optimum annealing temperature of quadruple PCR was 58℃, the optimum ratio for final con-centration (μmol/L ) of primers was 0 . 1∶0 . 2∶0 . 2∶0 . 2 for CruA∶P-CaMV 35S∶T-CaMV 35S∶pat, the sensitivity of the method was 0.01 ng/μL, and the amplified products of all the samples were completely consistent with the expectant molecular characteristics. ConclusionThe established quadruple PCR method for detecting herbicide-resistant transgenic rapeseed T45 can detect simultaneously endogenous gene and multiple exogenous genes of herbicide-resistant transgenic rapeseed T45 in a reaction system, so which can used as another efficient method to detect transgenic rapeseed T45.

     

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