转基因大豆DAS44406-6多重荧光定量PCR检测方法的建立

本文针对大豆内源基因Lectin和转基因大豆DAS44406-6品系的5’端插入位点序列,设计特异性引物及探针,建立同时检测转基因大豆DAS44406-6品系和大豆内源基因Lectin的多重荧光定量PCR方法,并运用15种转基因大豆、3种转基因玉米、1种转基因油菜、1种转基因水稻和非转基因大豆对该方法进行特异性评价,并分析该方法的灵敏度和稳定性。结果显示,该方法能准确从20种转基因样品和1种非转基因样品中检出靶目标,检测结果与待检样品信息一致,表明本方法具有良好的特异性;灵敏度高达0.01%;重复性实验表明DAS81419品系4种含量、9次重复反应Ct值的标准偏差介于0.050⁰.222,相对标准偏差介于0.169%⁰.677%,均在可接受范围内。该方法特异性强、灵敏度高、稳定性强,适用于各口岸实验室进行转基因大豆DAS44406-6的快速、准确的检测。 

Multiplex real- time PCR for the detection of genetically modified soybean DAS44406-6

Specific primers and probes based on the 5' flanking sequence of exogenous fragments of genetically modified( GM)soybean DAS44406-6 and endogenous Lectin gene of soybean were designed. A multiplex real- time PCR for the detection of GM soybean DAS44406- 6 and endogenous Lectin gene of soybean simultaneously had been developed. The specificity,sensitivity and stability of this method had been tested by 15 GM soybean,3 GM maize,1 GM rape,1 GM rice and 1 non- GM soybean.Results indicated that this multiplex real- time PCR method could accurately detect target from 20 GM samples and 1non- GM sample,which was in accordance to the predicted results..The sensitivity of this method was 0.01%.The repeatability test showed that the standard deviation( SD) of the Ct value ranged from 0.050 to 0.222 and the relative standard deviation( RSD) ranged from 0.169% to 0.677% of the 9 replicates about 4 concentration DNA templates,which were all in acceptable range.In conclusion,this multiplex real- time PCR method was high specific,sensitive and reliable,and would be an effective tool for the detection of GM DAS44406-6 in the laboratory of CIQ( China entry- exit inspection and quarantine bureau,CIQ).