利用多重PCR技术同时检测6种棉花转化体的方法研究

【目的】建立1种转基因棉花多重聚合酶链式反应(Polymerase chain reaction,PCR)检测方法。【方法】试验选择6种转基因棉花作为多重PCR检测对象,通过引物终浓度配比和引物退火温度的两要素全组合优化多重PCR反应体系,并分析方法灵敏度。【结果】多重PCR较优的MON1445、GHB614、MON15985、MON88913、LLCOTTON25、MON531引物终浓度为0.25、0.30、0.25、0.16、0.30、0.20μmol·L-1,较优引物退火温度为56℃,方法灵敏度为66个拷贝棉花基因组。利用盲样对上述体系的验证结果显示,所有盲样扩增条带与其含有的转基因转化体完全一致。【结论】本研究建立的体系可用于6种棉花转化体的快速检测。

Development of a Multiplex Polymerase Chain Reaction Method for Simultaneous Detection of Six Events in Genetically Modified Cotton

[Objective] The aim of this study was to establish a multiplex polymerase chain reaction (PCR) detection method for transgenic cotton.[Method] Six events in genetically modified cotton were selected for multiplex PCR.Specific primers were designed based on national standards or related literature.The ratio of primer concentration,annealing temperature,and sensitivity of the multiplex PCR reaction system were optimized.[Result] The optimal concentrations for primers MON1445,GHB614,MON 15985,MON88913,LLCOTTON25 and MON531 were 0.25,0.30,0.25,0.16,0.30,and 0.20 μmol·L-1,respectively;the optimal annealing temperature of the multiplex PCR was 56 ℃;the detection sensitivity of the method was 66 copies of the cotton genome.The verification results of the system showed that the amplified bands of all the known samples were identical to those of the transgenic events.[Conclusion] The system established in this study can be used for the rapid detection of six events occurring in genetically modified cotton.