应用多重PCR-液相悬浮芯片技术检测转基因水稻品系  

Development of Multiplex PCR Coupled with Liquid Bead Array for the Detection of Nine Genetically Modified Rice Events

作　　者：黄文胜[1] 傅凯[1] 邓婷婷[1] 李富威[1] 刘昊[1] 陈颖[1] 

机构地区：[1]中国检验检疫科学研究院,北京100123

出　　处：《食品科学》2014年第20期158-163,共6页Food Science

基　　金：国家科技重大专项(2014ZX08012-001);国家质检总局科技计划项目(2014IK083)

摘　　要：开展转基因水稻的多重聚合酶链式反应(polymerase chain reaction,PCR)液相芯片检测方法研究。针对科丰6号(KF6)、科丰8号(KF8)、华恢1号(BT63)、克螟稻(KMD1)、M12、T1C-19、T2A-1、LL62和LL601九种转基因水稻的侧翼序列,设计合成了在生物素标记的多重PCR扩增引物与固定在荧光编码微球上的探针,建立了2个多重PCR-液相芯片检测体系,可同时检测出这9种转基因水稻成分。结果表明,9种转基因水稻的引物和探针都具有较高的特异性,各组引物/探针之间无交叉扩增和非特异杂交,且在多重PCR-液相芯片检测中9种转基因水稻品系的相对检测灵敏度达到0.1%水平,符合欧盟和其他国家有关转基因产品标识的要求。本方法的检测效率和准确性均高于传统方法,可作为进出口转基因产品和国内转基因检测的有效方法。A multiplex PCR assay coupled with liquid bead array was developed for simultaneous identification of nine genetically modified rice(Oryzae sativa) lines. Based on the junction sequences between host plant genome DNA and the exogenous gene of the GM rice events(BT63, KF6, KF8, M12, KMD1, T1C-19, T2A-1, LL601, and LL62), the eventspecific primers and probes were designed for each event. In addition, in order to reduce the number of PCR reactions, two multiplex PCR systems were adopted and optimized by adjusting the concentration of each primer. Detection specificity was provided by capture probes designed for each GMO rice event which were covalently attached to florescent coding beads. The PCR amplicons were biotin-labeled and directly hybridized with the capture probes on the beads, and then the beads were detected according to protocol of Bio-Rad. The sensitivity of the assay was evaluated and the results indicated that the limit of detection was around 0.1% for 9 events. With improved detection efficiency and accuracy, the detection system complied with the requirements of current regulations in EU, Japan and China for the thresholds for the labeling of GMO, and could be applied in the enforcement of GM regulation in China.

关 键 词：多重PCR 液相芯片 转基因水稻 检测 

分 类 号：S511[农业科学—作物学]