转基因玉米GA21品系LAMP检测引物的筛选及方法的建立

Development of Loop-mediated Isothermal Amplification Method for Detection of Genitically Modified Maize Line GA21

根据转基因玉米GA21品系的特异序列设计了3套环介导等温扩增(LAMP)引物。通过反应温度优化、特异性测定,筛选到1套特异性好的LAMP引物,包括内引物、外引物和环引物各1对。在LAMP反应体系中加入SYBR Green I荧光染料,在实时荧光定量PCR仪上进行实时监测,对筛选到的LAMP引物进行反应条件、反应体系的优化,建立了转基因玉米GA21品系LAMP检测方法。对该方法的特异性、灵敏度、稳定性进行评价,结果表明建立的LAMP检测方法能特异、灵敏、稳定地检测转基因玉米GA21品系,检测限达到0.05%。将建立的LAMP检测方法应用于玉米及其制品中GA21品系的检测,检测结果与实时荧光PCR法的结果完全一致。

According to the specific sequence of genetically modified maize line GA21, 3 sets of primers for the Loop-mediated isothermal amplification（LAMP） were designed. 1 set of primers specific to LAMP were screened, including each 1 pair of the inner, outer and loop primers, through optimizating of reaction temperature and determinating of specificity. The LAMP method for detection of genetically modified maize GA21 was developed after optimizating of reaction conditions and system by real-time monitoring with SYBR Green I fluorescent dye added into the LAMP reaction system on the real-time fluorescent quantitative PCR instrument. The specificity, sensitivity and stability of the method was evaluated. The results showed that the LAMP method could detect specifically, sensitively and stably of transgenic corn GA21 strain with low detection limit of 0.05%. The detection results of GA21 strain in maize and its products by the LAMP method were same as those of the real-time fluorescent PCR.