A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize
A multiplex PCR method of detecting recombinant DNAs from five lines of genetically modified maize
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DOI：
10.3358/shokueishi.42.24
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发表时间：
2001-02-01
期刊：
影响因子：
--
通讯作者：
Hino, A
中科院分区：
文献类型：
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作者：
Matsuoka, T;Kuribara, H;Hino, A
日本已批准从美国进口七种转基因玉米作为食品和饲料。为了区分转基因玉米的五个品系，我们改进了先前报道的多重PCR方法。采用硅胶旋转柱试剂盒从转基因玉米中提取基因组DNA，可以缩短实验时间，提高实验室和环境中的安全性。对导入转基因玉米的重组DNA进行测序，并重新设计引物，提高PCR的特异性，利用多重PCR技术对5个转基因玉米品系进行区分。还设计了玉米内在玉米醇溶蛋白基因（Ze 1）的引物对，以确认可降解玉米DNA的存在。这6对引物扩增产物的长度不同。在同一试管中对5个转基因玉米品系的Ze 1和r-DNA进行了定性检测。特异性PCR条带可根据预期长度相互区分。从含有5个转基因玉米品系中的每一个的0.5%的玉米样品中可以检测到r-DNA。灵敏度对于确保非转基因材料的验证和监测标签系统的可靠性是可接受的。
Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.