改良LAMP法检测转基因作物

Detection of transgenic crops using improved loop-mediated isothermal amplification method

中文关键词:  LAMP  转基因作物  Bst DNA聚合酶  CaMV35S启动子  NOS终止子

英文关键词:loop-mediated isothermal amplification  genetically modified crops  Bst DNA polymerase  CaMV35S promoter  NOS terminator

中文摘要:

      目的 建立简易的可视化环介导等温扩增(LAMP)检测转基因作物的方法。方法 通过一对特异的能识别一段正义链和一段反义链的内引物和一对链置换引物, 在Bst DNA聚合酶作用下, 以等温条件实现对外源转基因成分的扩增, 反应液和高浓度的显色液SYBR GREENⅠ同时置于特殊设计的反应管中, 反应后可实现闭管可视化检测。结果 对转基因作物中常见的外源基因元件胭脂碱合成酶终止子(T-NOS)和烟草花叶病毒35S启动子(P-CaMV35S)进行了LAMP检测。T-NOS LAMP对大米品系BT63, 玉米品系BT11, MON810, 大豆品系GTS40-3-2的检测结果呈阳性, P-CaMV35S LAMP对大米品系BT63, 玉米品系BT11, MON810, MON863, 大豆品系GTS40-3-2的检测结果呈阳性, 二者对非转基因大米, 玉米, 大豆的检测结果均呈阴性。上述结果表明, 本文中建立的T-NOS 和P-CaMV35S LAMP检测方法特异性高。另外, 通过对大豆品系GTS40-3-2模拟样品的检测, 证明T-NOS LAMP能检出转基因成分含量为0.5%的样品, P-CaMV35S LAMP能检出转基因成分含量为0.1%的样品, 检测灵敏度较高。结论 改良LAMP法灵敏度高, 特异性好, 能用于快速初筛转基因作物。

英文摘要:

      Objective To establish a simple visual loop-mediated isothermal amplification (LAMP) detection method for genetically modified (GM) crops. Methods Under an isothermal condition, Bst DNA polymerase amplified the target sequences utilizing a pair of inner primers that contained sequences of sense and antisense strands of target DNA and a pair of displacement primers. The reaction mixture and a high concentration of fluorescent dye SYBR GREENⅠ were placed together in a specially designed reaction tube and the reaction results could be visually detected in the closed tube. Results In this experiment, the exogenous elements nonpaline synthase terminator (T-NOS) and tobacco mosaic virus 35S promoter (P-CaMV35S), which are common in GM crops, were detected by LAMP method. Rice event BT63, maize events BT11, MON863 and soybean event GTS40-3-2 gave T-NOS positive results, rice event BT63, maize event BT11, MON810, MON863 and soybean event GTS40-3-2 showed P-CaMV35S positive results. The T-NOS LAMP and P-CaMV35S LAMP results of non-GM rice, maize and soybean were negative. These results indicate that the T-NOS and P-CaMV35S LAMP detection methods established in this paper were highly specific. In addition, through the detection of GM soybean event GTS40-3-2 simulation samples, it was shown that the sensitivity of T-NOS LAMP was 0.5%, and the sensitivity of P-CaMV35S LAMP was 0.1%. The detection sensitivity was high. Conclusion The improved LAMP method has high sensitivity and good specificity, which can be used for rapid screening of GM crops.