NewLeaf-Y^(TM)转基因马铃薯PCR检测鉴定方法的建立

Establishment of PCR method for NewLeaf-Y^(TM ) GM potato detection and identification

建立了商业化种植的NewLeaf-YTM转基因马铃薯的筛选检测和鉴定的PCR方法。该方法根据马铃薯自身patatin基因作为内源特异参照基因扩增216bp片段,检查模板DNA提取的质量,避免了假阴性结果;同时扩增FMV35S启动子基因225bp片段和PVY-cp基因161bp片段。PCR反应循环参数是94℃2min;94℃40s,55℃60s,72℃60s,35次循环;之后72℃延伸5min。本实验中的F-primer(PVY01-5')/R-primer(PVY01-3')引物是针对商业化种植的NewLeaf-YTM转基因马铃薯PVY-cp抗病毒基因而设计的,具有很强的特异性,可以达到对NewLeaf-YTM转基因马铃薯鉴定的目的。

PCR method was established to detect and identify NewLeaf-Y TM GM potato.A set of commonly used primers of patatin sequence of potato were designed as endogenuous reference gene,and check on the quality of template extraction from potato,and avoid the result of false negative.One primers was designed based on FMV35S promoter with a amplicon of225bp,and other primers was designed based on PVY-cp with a amplicon of161bp.PCR amplification condition was one cycle of96℃for2min,then35cycle of(94℃for40s,60℃for50s,72℃for60s),and finally one cycle of72℃for5min.The primers of F-primer(PVY01-5')/R-primer(PVY01-3')was de-signed based on PVY-cp gene for commercial NewLeaf-Y TM GM potato.The method was specific for the identifica-tion of NewLeaf-Y TM GM potato.