大米和米制品Bt63转基因检测PCR方法的灵敏度研究.docx

浙江农业学报Acta Agriculturae 浙江农业学报Acta Agriculturae Zhejiangensis 21(6)：549-554，2009 大米和米制品Bt63转基因检测PCR方法的灵敏度研究 吴志毅，张明哲，陈 曦 (浙江出入境检验检疫局，浙江杭州310012) 摘要：采用普通PCR法和荧光定量PCR法对Bt63转基因大米的检测灵敏度进行对比研究。根据大米内源 蔗糖磷酸合成酶笨凶SIS和结构特异性基因Btc的基因序列，选用特异性的引物和探针进行研究，经验证试 验表明具有专一性，能用于品系鉴定。在灵敏度试验中。阳性转基因大米按照不同质量百分比进行梯度稀释， 提取DNA进行PCR扩增。结果表明，普通PCR检测的灵敏度在0．1％左右，而荧光定量PCR检测的灵敏度为 0．01％．是普通PCR检测的10倍以上。应用两种方法对实际样品进行检测，都得到了较明显的内源S庵基因 的扩增．而Btc基圉都是阴性。荧光定鼋PCR方法避免了电泳检测的步骤，具有更高的安全性和简便性，因此 日常检测中，利用荧光定量PCR技术检测Bt63转基因大米及其产品是一种快速、灵敏和准确的检测手段。 关键词：转基因检测；普通PCR；荧光定量PCR；Bt63大米 中图分类号：髑207．3 文献标识码：A 文章编号：1004—1524(2009)06一0549—06 Study Oil the sensitivity of PCR detection for Bt63 rice and its products WU Zhi．yi．ZHANG Ming．zhe，CHEN Xi (Zhejiang Entry-Exit Inspection and Quarantine Bureau，Hangzhou 310012，China) Abstract：Comparative studies performed the sensitivity of general real-time PCR analyze Bt63 genetically modified rice．Based the DNA sequences of rice endogenous gene SPS and structure-specific gene Btc，we selected specific primem and fluorescent probes in attempt identify Bt63 rice．The results showed good specificity which could be capable of identifying various species．In the sensitivity assay，Bt63 rice Was serially di— luted according their mass percentage and then DNA Was extracted for PCR amplification．As result，fluorescent quantative PCR displayed tenfold higher sensitivity than general PCR．whose sensitivity values 0．1％and 0．01％， respectively．Two types of PCR methods applied analyze the food samples，both of which turned be good for effective amplification of SPS gene，while Btc gene retained negative．The real—time PCR method avoids the process of gel electrophoresis，with higher security and simplicity，therefore，in actual daily test，real—time PCR detection of Bt63 genetically modified rice and its products fast，sensitive and testing method． Key words：GMO detection；general PCR；real-time PCR；Bt63 rice 自从1983年世界上第一种转基因作物问世 主要有两个方面：一是对人体健康是否有害；二 以来，对转基因食品的安全性问题各界一