转基因玉米品系及转化体成分四重实时荧光PCR快速鉴定

Bt11转基因玉米品系具有抗草铵膦除草剂,鳞翅目昆虫抗性的耐除草剂抗虫玉米,MIR162和Mon89034是鳞翅目昆虫抗性的单抗虫玉米,均是国内外出入境监管主要关注的转基因玉米品系。本研究通过靶标基因筛选,转基因阳性样品采集,核酸样本制备,多重引物和荧光探针组合筛选,反应体系优化以及方法学验证等过程开发建立了四重荧光定量PCR检测技术。结果表明该技术的使用可实现一个反应管中同时检测MIR162、Bt11、Mon89034三个玉米品系的特异性基因序列和一个编码玉米淀粉合成酶异构体zSTSII-2 (zSSIIb) 玉米内源基因。通过阳性对照,阴性对照和空白对照特异性S曲线与对应的阈值大小分析可判定样品中是否含有这三个转基因玉米品系及其转化体成分。经方法学特异性检测,结果与转基因检测金标准的单实时荧光PCR结果一致；经平行样和灵敏度测试,最低检测限为18个拷贝；经标准曲线扩增分析,四个基因的扩增效率均在90%~110%范围内,扩增效果良好。该方法从DNA提取到报告结果不足3小时,可缩短检测时限,节约试剂耗材成本,操作简单易行,满足高通量特征,可为市场流通产品品系和转基因成分的实时监管和快速鉴定提供技术支撑。

Rapid Identification of Transgenic Maize Lines and Transformants by Quadruplex Real-time PCR

Transgenic maize Bt11 have herbicide resistance and insect resistance to glufosinate herbicide and Lepidoptera insects. MIR162 and MON89034 are single insect resistant with Lepidoptera insect resistance, which are the transgenic maize lines mainly concerned by domestic and foreign supervision. In this study, we developed and established a quadruple fluorescent quantitative PCR detection technology through target gene screening, transgenic positive sample collection, nucleic acid sample preparation, multiple primer and fluorescent probe combination screening, reaction system optimization and methodology validation. The results showed that the specific gene sequences of MIR162, Bt11 and MON89034 and an endogenous gene which is Coding for Zea mays starch synthase isomer zSTSII-2 (zSSIIb) could be detected simultaneously in a reaction tube by using this technique. The specific amplification curve of positive control, negative control and blank control and the corresponding threshold value could be used to determine whether the samples contained the three transgenic maize lines and their transformants. The results were consistent with the results of single real-time fluorescent PCR in the common standard of transgenic detection; the detection limit was 18 copies by parallel sample and sensitivity test; the amplification efficiency of the four genes was in the range of 90% ~ 110% by standard curve amplification analysis, and the amplification efficiency was good. The method is less than 3 hours from DNA extraction to report results, which can shorten the detection time limit, save the cost of reagent, and is simple and easy to operate, meeting the high-throughput characteristics. It can provide technical support for the real-time supervision and rapid identification of market products and genetically modified ingredients.